Prevention and treatment of verotoxin-induced disease

ABSTRACT

The present invention includes methods for generating neutralizing antitoxin directed against verotoxins. In preferred embodiments, the antitoxin directed against these toxins is produced in avian species using soluble recombinant verotoxin proteins. This antitoxin is designed so as to be administrable in therapeutic amounts and may be in any form (i.e., as a solid or in aqueous solution). These antitoxins are useful in the treatment of humans and other animals intoxicated with at least one bacterial toxin, as well as for preventive treatment, and diagnostic assays to detect the presence of toxin in a sample.

FIELD OF THE INVENTION

[0001] The present invention relates to antitoxin therapy and preventionof disease due to Escherichia coli verotoxin in humans and otheranimals, and diagnostic assays to detect toxins. In particular, thepresent invention relates to the isolation of polypeptides derived fromEscherichia coli verotoxins, and the use thereof as immunogens for theproduction of vaccines, including multivalent vaccines, and antitoxins.

BACKGROUND OF THE INVENTION

[0002] A. Escherichia coli as a Pathogenic Organism

[0003]Escherichia coli is the organism most commonly isolated inclinical microbiology laboratories, as it is usually present as normalflora in the intestines of humans and other animals. However, it is animportant cause of intestinal, as well as extraintestinal infections.For example, in a 1984 survey of nosocomial infections in the UnitedStates, E. coli was associated with 30.7% of the urinary tractinfections, 11.5% of the surgical wound infections, 6.4% of the lowerrespiratory tract infections, 10.5% of the primary bacteremia cases,7.0% of the cutaneous infections, and 7.4% of the other infections (J.J. Farmer and M. T. Kelly, “Enterobacteriaceae,” in Manual of ClinicalMicrobiology, Balows et al.(eds), American Society for Microbiology,[1991], p. 365). Surveillance reports from England, Wales and Irelandfor 1986 indicate that E. coli was responsible for 5,473 cases ofbacteremia (including blood, bone marrow, spleen and heart specimens);of these, 568 were fatal. For spinal fluid specimens, there were 58cases, with 10 fatalities (J. J. Farmer and M. T. Kelly,“Enterobacteriaceae,” in Manual of Clinical Microbiology, Balows etal.(eds), American Society for Microbiology, [1991], p. 366 ). There areno similar data for United States, as these are not reportable diseasesin this country.

[0004] Studies in various countries have identified certain serotypes(based on both the O and H antigens) that are associated with the fourmajor groups of E. coli recognized as enteric pathogens. Table 1 listscommon serotypes included within these groups. The first group includesthe classical enteropathogenic serotypes (“EPEC”); the next groupincludes those that produce heat-labile or heat-stable enterotoxins(“ETEC”); the third group includes the enteroinvasive strains (“EIEC”)that mimic Shigella strains in their ability to invade and multiplywithin intestinal epithelial cells; and the fourth group includesstrains and serotypes that cause hemorrhagic colitis or produceShiga-like toxins (or verotoxins) (“VTEC” or “EHEC” [enterohernmorrhagicE. coli ]). TABLE 1 Pathogenic E. coli Serotypes Group AssociatedSerotypes Entero- O6:H16; O8:NM; O8:H9; O11:H27; O15:H11; O20:NM;toxigenic O25:NM; O25:H42; O27:H7; O27:H20; O63:H12; O78:H11; (ETEC)O78:H12; O85:H7; O114:H21; O115:H21; O126:H9; O128ac:H7; O128ac:H12;O128ac:H21; O148:H28; O149:H4; O159:H4; O159:H20; O166:H27; and O167:H5Entero- O26:NM; O26:H11; O55:NM; O55:H6; O86:NM; O86:H2; pathogenicO86:H34; O111ab:NM; O111ab:H2; O111ab:H12; (EPEC) O111ab:H21; O114:H2;O119:H6; O125ac:H21; O127:NM; O127:H6; O127:H9; O127:H21; O128ab:H2;O142:H6; and O158:H23 Entero- O28ac:NM; O29:NM; O112ac:NM; O115:NM;O124:NM; invasive O124:H7; O124:H30; O135:NM; O136:NM; O143:NM; (EIEC)O144:NM; O152:NM; O164:NM; and O167:NM Verotoxin- O1:NM; O2:H5; O2:H7;O4:NM; O4:H10; O5:NM; O5:H16; Producing O6:H1; O18:NM; O18:H7; O25:NM;O26:NM; O26:H11; (VTEC)) O26:H32; O38:H21; O39:H4; O45:H2; O50:H7;O55:H7; O55:H10; O82:H8; O84:H2; O91:NM; O91:H21; O103:H2; O111:NM;O111:H8; O111:H30; O111:H34; O113:H7; O113:H21; O114:H48; O115:H10;O117:H4; O118:H12; O118:H30; O121:NM; O121:H19; O125:NM; O125:H8;O126:NM; O126:H8; O128:NM; O128:H2; O128:H8; O128:H12; O128:H25;O145:NM; O125:H25; O146:H21; O153:H25; O157:NM; O157:H7; O163:H19;O165:NM; O165:19; and O165:H25

[0005] B. Verotoxin Producing Strains of E. coli

[0006] Although all of these disease-associated serotypes causepotentially life-threatening disease, E. coli 0157:H7 and otherverotoxin-producing strains have recently gained widespread publicattention in the United States due to their recently recognizedassociation with two serious extraintestinal diseases, hemolytic uremicsyndrome (“HUS”) and thrombotic thrombocytopenic purpura (“TTP”).Worldwide, E. coi 0157:H7 and other verotoxin-producing E. coli (VTEC)are an increasingly important human health problem. First identified asa cause of human illness in early 1982 following two outbreaks offood-related hemorrhagic colitis in Oregon and Michigan (M. A. Karmali,“Infection by Verocytotoxin-Producing Escherichia coli,” Clin.Microbiol. Rev., 2:15-38 [1989]; and L. W. Riley, et al. “Hemorrhagiccolitis associated with a rare Escherichia coli serotype,” New Eng. J.Med., 308: 681-685 [1983]), the reported incidence of VTEC-associateddisease has risen steadily, with outbreaks occurring in the U.S.,Canada, and Europe.

[0007] With increased surveillance, E. coli 0157:H7 has been recognizedin other areas of the world including Mexico, China, Argentina, Belgium,and Thailand (N. V. Padhye and M. P. Doyle, “Escherichia coli O157:H7:Epidemiology, pathogenesis and methods for detection in food,” J. Food.Prot., 55: 555-565 [1992]; and P. M. Griffin and R. V. Tauxe, “Theepidemiology of infections caused by Escherichia coli 0157:H7, otherenterohemorrhagic E. coli , and the associated hemolytic uremicsyndrome,” Epidemiol. Rev., 13: 60 [1991]).

[0008] The disease attracted national attention in the U.S. after amajor outbreak in the Pacific Northwest that was associated withconsumption of undercooked E. coli O157:H7-contaminated hamburgers. Over700 hundred people fell ill (more than 170 were hospitalized) and fouryoung children died (P. Recer, “Experts call for irradiation of meat toprotect against food-borne bacteria,” Associated Press, Jul. 12. 1994[1994]). Several outbreaks since then have underscored the potentialseverity and multiple mechanisms for transmission of VTEC-associateddiseases (M. Bielaszewska et al., “Verotoxigenic (enterohaemorrhagic)Escherichia coli in infants and toddlers in Czechoslovakia,” Infection18: 352-356 [1990]; A. Caprioli et al., “Hemolytic-uremic syndrome andVero cytotoxin-producing Escherichia coli infection in Italy, “J.Infect. Dis., 166: 184-158 [1992]; A. Caprioli, et al., “Community-wideOutbreak of Hemolytic-Uremic Syndrome Associated with Non-O157Verocytotoxin-Producing Escherichia coli,” J. Infect. Dis., 169: 208-211[1994]; N. Cimolai, “Low frequency of high level Shiga-like toxinproduction in enteropathogenic Escherichia coli serogroups,” Eur. J.Pediatr., 151: 147 [1992]; and R. Voelker., “Panel calls E. coliscreening inadequate,” Escherichia coli O157:H7—Panel sponsored by theAmerican Gastroenterological Association Foundation in July 1994,Medical News & Perspectives, J. Amer. Med. Assoc., 272: 501 [1994]).

[0009] While O157:H7 is currently the predominant E. coli serotypeassociated with illness in North America, other serotypes (as shown inTable 1, and in particular 026:H11, 0113:H21, 091:H21 and O11:NM) alsoproduce verotoxins which appear to be important in the pathogenesis ofgastrointestinal manifestations and the hemolytic uremic syndrome (P. M.Griffin and R. V. Tauxe, “The epidemiology of infections caused byEscherichia coli O157:H7, other enterohemorrhagic E. coli , and theassociated hemolytic uremic syndrome,” Epidemiol. Rev., 13: 60 [1990];M. M. Levine, et al., “Antibodies to Shiga holotoxin and to twosynthetic peptides of the B subunit in sera of patients with Shigelladysenteriae 1 dysentery,” J. Clin. Microbiol., 30: 1636-1641 [1992]; andC. R. Dorn, et al., “Properties of Vero cytotoxin producing Escherichiacoli of human and animal origin belonging to serotypes other thanO157:H7,” Epidemiol. Infect., 103: 83-95 [1989]). Since organisms withthese serotypes have been shown to cause illness in humans they mayassume greater public health importance over time (P. M. Griffin and R.V. Tauxe, ” The epidemiology of infections caused by Escherichia coliO157:H7, other enterohemorrhagic E coli, and the associated hemolyticuremic syndrome,” Epidemiol. Rev., 13: 60 [1990]).

[0010] Clinicians usually observe cases of hemolytic uremic syndrome(“HUS”) clustered in a geographic region. However, small outbreaks arelikely to be missed because many laboratories do not routinely screenstool specimens for E. coli O157:H7. Many cases related tonon-commercial food preparation also probably go unrecognized.Nonetheless, E. coli O157:H7 is responsible for a large number of cases,as more than 20,000 cases of E. coli O157:H7 infection are reportedannually in the U.S., with 400-500 deaths from HUS. However, theseestimates were compiled when only 11 states mandated reporting of E.coli O157:H7. Twenty-nine states have recently made E. coli O157:H7infection a reportable disease (R. Voelker, “Panel calls E. coliscreening inadequate; Escherichia coli O157:H7; panel sponsored by theAmerican Gastroenterological Association Foundation in July 1994,Medical News & Perspectives,” J. Amer. Med. Assoc., 272: 501 [1994]).Indeed, the Centers for Disease Control recently added E coli O157:H7 totheir list of reportable diseases (“Public Health Threats,” Science267:1427 [1995]).

[0011] C. Nature of Verotoxin-Induced Disease

[0012] Risk factors for HUS progression following infection with E. coliO157:H7 include age (very young or elderly), bloody diarrhea,leukocytosis, fever, large amounts of ingested pathogen, previousgastrectomy, and the use of antimicrobial agents (in particular,trimethoprim-sulfamethoxazole)(A. A. Harris et al., “Results of ascreening method used in a 12 month stool survey for Escherichia coliO157:H7,” J. Infect. Dis., 152: 775-777 [1985]; and M. A. Karmali,“Infection by Verocytotoxin-producing Escherichia coli,” Clin.Microbiol. Rev., 2: 15-38 [1989]).

[0013] As indicated above, E. coli O157:H7 is associated withsignificant morbidity and mortality. The spectrum of illness associatedwith E. coli O157:H7 infection includes asymptomatic infection, milduncomplicated diarrhea, hemorrhagic colitis, HUS, and TTP″. Hemorrhagiccolitis (or “ischemic colitis”) is a distinct clinical syndromecharacterized by sudden onset of abdominal cramps-likened to the painassociated with labor or appendicitis-followed within 24 hours by waterydiarrhea. One to two days later, the diarrhea turns grossly bloody inapproximately 90% of patients and has been described as “all blood andno stool” (C. H. Pai et al., “Sporadic cases of hemorrhagic colitisassociated with Escherichia coli O157:H7,” Ann. Intern. Med., 101:738-742 [1984]; and R. S. Remis et al., “Sporadic cases of hemorrhagiccolitis associated with Escherichia coli O157:H7,” Ann. Intern. Med.,101: 738-742 [1984]). Vomiting may occur, but there is little or nofever. The time from ingestion to first loose stool ranges from 3-9 days(with a mean of 4 days) L. W. Riley et al., “Hemorrhagic colitisassociated with a rare Escherichia coli serotype,” New Eng. J. Med.,308: 681-685 [1983]; and D. Pudden et al., “Hemorrhagic colitis in anursing home,” Ontario Can. Dis. Weekly Rpt., 11: 169-170 [1985]), andthe duration of illness ranges generally from 2-9 days (with a mean of 4days).

[0014] HUS is a life-threatening blood disorder that appears within 3-7days following onset of diarrhea in 10-15% of patients. Those youngerthan 10 years and the elderly are at particular risk. Symptoms includerenal glomerular damage, hemolytic anemia (rupturing of erythrocytes asthey pass through damaged renal glomeruli), thrombocytopenia and acutekidney failure. Approximately 15% of patients with HUS die or sufferchronic renal failure. Indeed, HUS is a leading cause of renal failurein childhood (reviewed by M. A. Karmali, “Infection byVerocytotoxin-producing Escherichia coli,” Clin. Microbiol. Rev., 2:15-38 [1989]). Currently, blood transfusion and dialysis are the onlytherapies for HUS.

[0015] TTP shares similar histopathologic findings with HUS, but usuallyresults in multiorgan microvascular thrombosis. Neurological signs andfever are more prominent in TTP, compared with HUS. Generally occurringin adults, TTP is characterized by microangiopathic hemolytic anemia,profound thrombocytopenia, fluctuating neurologic signs, fever and mildazotemia (H. C. Kwaan, “Clinicopathological features of thromboticthrombocytopenic purpura,” Semin. Hematol., 24: 71-81 [1987]; and S. J.Machin, “Clinical annotation: Thrombotic thrombocytopenic purpura,” Br.J. Hematol., 56: 191-197 [1984]). Patients often die from microthrombiin the brain. In one review of 271 cases, a rapidly progressive coursewas noted, with 75% of patients dying within 90 days (E. L. Amorosi andJ. E. Ultmann, “Thrombotic thrombocytopenic purpura: Report of 16 casesand review of the literature,” Med., 45:139-159 (1966).

[0016] Other diseases associated with E. coli O157:H7 infection includehemorrhagic cystitis and balantitis (W. R. Grandsen et al., “Hemorrhagiccystitis and balantitis associated with verotoxin-producing Escherichiacoli O157:H7,” Lancet ii: 150 [1985]), convulsions, sepsis with otherorganisms and anemia (P. C. Rowe et al., “Hemolytic anemia afterchildhood Escherichia coli O157:H7 infection: Are females at increasedrisk?” Epidemiol. Infect., 106: 523-530 [1991]).

[0017] D. Mechanism of Pathogenesis

[0018] Verotoxins are strongly linked to E. coli O157:H7 pathogenesis.All clinical isolates of E. coli O157:H7 have been shown to produce oneor both verotoxins (VT1 and VT2) (C. A. Bopp et al., “UnusualVerotoxin-producing Escherichia coli associated with hemorrhagiccolitis,” J. Clin. Microbiol., 25: 1486-1489 [1987]). The VT1 and VT2genes are carried by temperate coliphages 933J and 933W, respectively.Once lysogenized. these coliphages lead to the expression of toxin genesby the E. coli host.

[0019] Both of these toxins are cytotoxic to Vero (African green monkeykidney) and HeLa cells, and cause paralysis and death in mice (A. D.O'Brien et al., “Purification of Shigella dysenteriae 1 (Shiga) liketoxin from Escherichia coli O157:H7 strain associated with hemorrhagiccolitis,” Lancet ii: 573 [1983]). These toxins are sometimes referred toin the literature as Shiga-like toxins I and II (SLT-I and SLT-II,respectively), due to their similarities with the toxins produced byShigella. Indeed, much of our understanding of E. coli VTs is based oninformation accumulated on Shiga toxins. Shiga toxin, first described in1903, has been recognized as one of the most potent bacterial toxins foreukaryotic cells (reviewed by M. A. Karmali, “Infection byVerocytotoxin-producing Escherichia coli,” Clin. Microbiol. Rev., 2:15-38 [1989]). Hereinafter, the VT convention will be used; thus, VT1and VT2 correspond to SLT-I and SLT-II, respectively.

[0020] While the pathogenic mechanism of E. coli O157:H7 infection isincompletely understood, it is believed that ingested organisms adhereto and colonize the intestinal mucosa, where toxins are released whichcause endothelial cell damage and bloody diarrhea. It is also postulatedthat hemorrhagic colitis progresses to HUS when verotoxins enter thebloodstream, damaging the endothelial cells of the microvasculature andtriggering a cascade of events resulting in thrombus deposition in smallvessels. These microthrombi occlude the microcapillaries of the kidneys(particularly in the glomeruli) and other organs, resulting in theirfailure (J. J. Byrnes and J. L. Moake, “TTP and HUS syndrome: Evolvingconcepts of pathogenesis and therapy,” Clin. Hematol., 15: 413-442[1986]; and T. G. Cleary, “Cytotoxin-producing Escherichia coli and thehemolytic uremic syndrome,” Pediatr. Clin. North Am., 35: 485-501[1988]). Verotoxins entering the bloodstream may also result in directkidney cytotoxicity.

[0021] VT1 is immunologically and structurally indistinguishable fromShiga toxin produced by Shigella dysenteriae (A. D. O'Brien et al.,“Purification of Shigella dysenteriae 1 (Shiga) like toxin fromEscherichia coli O157:H7 strain associated with hemorrhagic colitis,”Lancet ii: 573 [1983]). VT1 and VT2 holotoxins each consist of one A andfive B subunits (A. Donohue-Rolfe et al., “Purification of Shiga toxinand Shiga-like toxins I and II by receptor analog affinty chromatographywith immobilized P1 glycoprotein and production of cross reactivemonoclonal antibodies,” Infect. Immun., 57: 3888-3893 [1989]; and A.Donohue-Rolfe et al., “Simplified high yield purification of Shigellatoxin and characterization of subunit composition and function by theuse of subunit-specific monoclonal and polyclonal antibodies,” J. Exp.Med., 160: 1767-1781 [1984]). Intra-chain disulfide bonds are formed andthe holotoxin is assembled after secretion of the subunits to theperiplasm. Each subunit contains a leader sequence that targetssecretion of the toxin. VT1 and VT2 are structurally related, sharing56% amino acid homology.

[0022] The toxic A subunit is enzymatically active, while the B subunitbinds the holotoxin to the receptor on the target eukaryotic cell. The Achain is structurally related to the ricin A chain, and acts in asimilar manner to inhibit protein synthesis by cleaving a single adenineresidue from 28S ribosomal RNA (Endo et al., J. Biol. Chem.,262:5908-5912 [1987]). The A chain is 32 (VT1) or 33 (VT2) kd in size,and is proteolytically cleaved into Al (approximately 27 kd) and A2(approximately 3-4 kd) fragments. In both VT1 and VT2, the non-toxic Bsubunit is approximately 8 kd. Pentamers of the B subunit bind mammaliancell surface receptors, facilitating internalization of holotoxin bycells.

[0023] Crystal structure analysis of Shiga holotoxin and VT1 B subunitpentamers have shown that the holotoxin assembles with the C-terminalend of the A subunit associating with, and inserting within, a pentamerof B chains (P. E. Stein et al., “Crystal structure of the cell-bindingB oligomer of vertoxin-1 from E. coli ,” Nature 355: 748-750 [1992]; andM. E. Fraser et al., “Crystal structure of the holotoxin from Shigelladysenteriae at 2.5 Å resolution,” Struct. Biol., 1:59-64 [1994]). Thealpha helical C-terminal region of the A chain (residues 279-293) isencircled by a pentameric ring of B subunits, with the remainder of theA chain exposed. This conformation is consistent with the observationthat a C-terminally truncated A1 subunit of VT1 is toxic (in a ribosomalinhibition assay), but cannot associate with B subunit pentamers (P. R.Austin et al, “Evidence that the A₂ fragment of Shiga-like toxin type Iis required for holotoxin integrity,” Infect. Immun., 62: 1768 [1994]).

[0024] The Verotoxin A Subunit. Examination of the crystal structure ofShiga holotoxin indicates that the N-terminus of its A subunit is bothsurface-exposed and functionally important. Removal of amino acidinterval 3-18 of the A subunit completely abolished toxicity (L. P.Perera et al., “Mapping the minimal contiguous gene segment that encodesfunctionally active Shiga-like toxin II,” Infect. Immun., 59: 829-835[1991]) while removal of interval 25-44 retained toxicity but abolishedits association with B subunit pentamers (J. E. Haddad et al., “Minimumdomain of the Shiga toxin A subunit required for enzymatic activity,” J.Bacteriol., 175: 4970-4978 [1993]). Deletion of the first 13 residues ofthe homologous ricin A subunit also abolished toxicity, while deletionof the first 9 residues did not (M. J. May, et al., “Ribosomeinactivation by ricin A chain: A sensitive method to assess the activityof wild-type and mutant polypeptides,” EMBO J., 8: 301-308 [1989]).

[0025] The Verotoxin B Subunit. Studies of Shiga toxin B subunit suggestthat neutralizing epitopes may also be present at both the N- andC-terminal regions of VTl and VT2 B subunits. Polyclonal antibodiesraised against peptides from these regions (residues 5-18, 13-26, 7-26,54-67 and 57-67) show partial neutralization of Shiga toxin (I. Harariand R. Arnon, “Carboxy-terminal peptides from the B subunit of Shigatoxin induce a local and parenteral protective effect,” Mol. Immunol.,27: 613-621 [1990]; and I. Harari et al., “Synthetic peptides of Shigatoxin B subunit induce antibodies which neutralize its biologicalactivity,” Infect. Immun., 56: 1618-1624 [1988]). Deletion of the lastfive amino acids of Shiga toxin B (M. P. Jackson et al., “FunctionalAnalysis of the Shiga toxin and Shiga-like toxin Type II variant bindingsubunits by using site-directed mutagenesis,” J. Bacteriol., 172:653-658 [1990]), or four amino acids of VT2 B (L. P. Perera et al.,“Mapping the minimal contiguous gene segment that encodes functionallyactive Shiga-like toxin II,” Infect. Inunun., 59: 829-835 [1991]),eliminate toxin activity, while deletion of the last two amino acids ofVT2 B subunit reduced cytotoxicity. In contrast, the addition of an 18or 21 amino acid extension to the native C-terminus of the VT2 B subunitwas presumably conformationally correct, as these proteins assembledcytotoxic holotoxin.

[0026] Various approaches to express recombinant verotoxins haveincluded individual or coordinate expression of A and B subunits fromhigh-copy number plasmids and expression with fusion partners (J. E.Haddad et al., “Minimum domain of the Shiga toxin A subunit required forenzymatic activity,” J. Bacteriol., 175: 4970-4978; J. E. Haddad, and M.P. Jackson, “Identification of the Shiga toxin A-subunit residuesrequired for holotoxin assembly,” J. Bacteriol., 175: 7652-7657 [1993];M. P. Jackson et al., “Mutational analysis of the Shiga toxin andShiga-like toxin II enzymatic subunits,” J. Bacteriol., 172: 3346-3350[1990]; C. J. Hovde et al., “Evidence that glutamic acid 167 is anactive-site residue of Shiga-like toxin I,” Proc. Natl. Acad. Sci., 85:2568-2572 [1988]; R. L. Deresiewicz et al., “The role of tyrosine-1 14in the enzymatic activity of the Shiga-like toxin I A-chain,” Mol. Gen.Genet., 241: 467-473 [1993]; T. M. Zollman et al., “Purification ofRecombinant Shiga-like Toxin Type I A1 Fragment from Escherichia coli,”Protein Express. Purific., 5: 291-295 [1994]; K. Ramotar, et al.,“Characterization of Shiga-like toxin I B subunit purified fromoverproducing clones of the SLT-I B cistron,” Biochem J., 272: 805-811[1990]; S. B. Calderwood et al., “A system for production and rapidpurification of large amounts of the Shiga toxin/Shiga-like toxin I Bsubunit,” Infect. Immun., 58: 2977-2982 [1990]; D. W. K. Acheson, etal., “Comparison of Shiga-like toxin I B-subunit expression andlocalization in Escherichia coli and Vibrio cholerae by using trc oriron-regulated promoter systems,” Infect. Immun. 61: 1098-1104 [1993];M. P. Jackson et al., “Nucleotide sequence analysis and comparison ofthe structural genes for Shiga-like toxin I and Shiga-like toxin IIencoded by bacteriophages from Escherichia coli 933,” FEMS Microbiol.Lett., 44: 109-114 [1987]; J. W. Newland et al., “Cloning of genes forproduction of Escherichia coli Shiga-like toxin type II,” Infect. Immun.55: 2675-2680 [1987]; and F. Gunzer and H. Karch, “Expression of A and Bsubunits of Shiga-like toxin II as fusions with glutathioneS-transferase and their potential for use in seroepidemiology,” J. Clin.Microbiol., 31: 2604-2610 [1993]; and D. W. Acheson et al., “Expressionand purification of Shiga-like toxin II B subunits,” Infect. Immun.,63:301-308 [1995]). In one case, bench top fermentation techniquesyielded 22 mg/liter of soluble recombinant protein (D. W. K. Acheson, etal., “Comparison of Shiga-like toxin I B-subunit expression andlocalization in Escherichia coli and Vibrio cholerae by using trc orIron-regulated promoter systems,” Infect. Immun. 61: 1098-1104 [1993]).However, there have been no systematic approaches to identifying theappropriate spectrum of VT antigens, preserving immunogen andimmunoabsorbant antigenicity and scaling-up.

[0027] The receptor for VT1 and VT2 is a globotriaosyl ceramidecontaining a galactose α-(1-4)- galactose-β-(1-4) glucose ceramide (Gb3)(C. A. Lingwood et al., “Glycolipid binding of natural and recombinantEscherichia coli produced verotoxin in vitro,” J. Biol. Chem., 262:1779-1785 [1987]; and T. Wadell et al., “Globotriaosyl ceramide isspecifically recognized by the Escherichia coli verocytotoxin 2,”Biochem. Biophys. Res. Commun., 152: 674-679 [1987]). Gb3 is abundant inthe cortex of the human kidney and is present in primary humanendothelial cell cultures. Hence, the identification of Gb3 as thefunctional receptor for VT1 and VT2 is consistent with their role in HUSpathogenesis, in which endothelial cells of the renal vasculature arethe principal site of damage. Therefore, toxin-mediated pathogenesis mayfollow a sequence of B subunit binding to Gb3 receptors on kidney cells,toxin internalization, enzymatic reduction of the A subunit to an Alfragment, binding of the Al subunit to the 60S ribosomal subunit,inhibition of protein synthesis and cell death (A. D. O'Brien et al.,“Shiga and Shiga-like toxins. Microbial Rev., 51: 206-220 [1987]).

[0028] The role of verotoxins in the pathogenesis of E. coli O157:H7infections has been further studied in animal models. Infection or toxinchallenge of laboratory animals do not produce all the pathologies andsymptoms of hemorrhagic colitis, HUS, and TTP which occur in humans.Glomerular damage is noticeably absent. Nonetheless, experiments usinganimal models implicate verotoxins as the direct cause of hemorrhagiccolitis, microvascular damage leading to the failure of kidneys andother organs and CNS neuropathies.

[0029] For example, Barrett, et al. delivered VT2 into the peritonealcavity of rabbits using mini-osmotic pumps (J. J. Barrett et al.,“Continuous peritoneal infusion of shiga-like toxin II (SLTII) as amodel for SLT II-induced diseases,” J. Infect. Dis., 159: 774-777[1989]). In three days, most animals receiving the toxin developeddiarrhea, with intestinal lesions resembling those seen in humans withhemorrhagic colitis. Although there was some evidence of renaldysfunction, none of the rabbits developed HUS. Beery, et al. showedthat VT2, when administered intraperitoneally or intravenously to adultmice, produces lesions of the kidneys and colon (J. T. Beery et al.,“Cytotoxic activity of Escherichia coli O157:H7 culture filtrate on themouse colon and kidney,” Curr. Microbiol., 11: 335-342 [1984]).Histologic lesions in the kidney included accumulation of numerousexfoliated collecting tubules and marked intracellular vacuolation ofproximal convoluted tubular cells.

[0030] Sjogren et. al. studied the pathogenesis of an entero-adherentstrain of E. coli (RDEC-1) lysogenized with a VT1-containingbacteriophage (VT1-producing RDEC-1) (R. Sjogren et al., “Role ofShiga-like toxin I in bacterial enteritis: comparison between isogenicEscherichia coli strains induced in rabbits,” Gastroenterol., 106:306-317 [1994]). In this study, rabbits were challenged with RDEC-1 orVT1-producing RDEC-1 and studied for onset of disease. The VT1-producingvariant induced a severe, non-invasive, entero-adherent infection inrabbits which was characterized by serious histological lesions withvascular changes, edema and severe epithelial inflammation. Importantly,vascular changes consistent with endothelial damage were seen ininfected animals that was similar to intestinal microvascular changes inhumans with E. coli O157:H7 infection. Based on these observations, theyconcluded that VT1 is an important virulence factor in enterohemorrhagicE. coli O157:H7 infection.

[0031] Fuji et. al. described a model in which mice were treated forthree days with streptomycin followed by a simultaneous challenge of E.coli O157:H7 orally, and mitomycin intraperitoneally (J. Fuji et al.,“Direct evidence of neuron impairment by oral infection withVerotoxin-producing Escherichia coli O157:H7 in mitomycin-treated mice,”Infect. Immun., 62: 3447-34453 [1994]). All of the animals died withinfour days. Immunoelectron-microscopy strongly suggested that death wasdue to the toxic effects of VT2v (a structural variant of VT2), on boththe endothelial cells and neurons in the central nervous system whichresulted in fatal acute encephalopathy.

[0032] Wadolkowski et al. studied colonization of E. coli O157:H7 inmice. Mice were treated with streptomycin and fed 10¹⁰ E. coli O157:H7(E. A. Wadolkowski et al., “Mouse model for colonization and diseasecaused by enterohemorrhagic Escherichia coli O157:H7,” Infect. Immun.,58: 2438-2445 [1990]; and E. A. Wadolkowski et al., “Acute renal tubularnecrosis and death of mice orally infected with Escherichia coli strainsthat produce Shiga-like toxin Type II,” Infect. Immun., 58: 3959-3965[1990]). All of the mice died due to severe, disseminated, acutenecrosis of proximal convoluted tubules. In mouse models, glomerulardamage was not observed, but toxic acute renal tubular necrosis wasobserved which is characteristic of some HUS patients. The failure ofmice to show glomerular damage is thought to be due to the absence of afunctional globotriaosyl ceramide receptor specific for verotoxins inthe glomeruli of the kidneys. Administration of VT2 subunit-specificmonoclonal antibodies prior to infection prevented all pathology anddeath.

[0033] E. Current Therapeutic Approaches

[0034]E. coli O157:H7 disease is not adequately controlled by currenttherapy. Patient treatment is tailored to manage fluid and electrolytedisturbances, anemia, renal failure and hypertension. Although E. coliO157:H7 is susceptible to common antibiotics, the role of antibiotics inthe treatment of infection has questionable merit. In both retrospectiveand prospective studies, prophylaxis or treatment with antibiotics suchas trimethoprim-sulfamethoxazole, there was either no benefit or anincreased risk of developing HUS (T. N. Bokete et al., “Shiga-like toxinproducing Escherichia coli in Seattle children: a prospective study,”Gastroenterol., 105: 1724-1731 [1993]; A. T. Pavia et al., “Hemolyticuremic-syndrome during an outbreak of Escherichia coli O157:H7infections in institutions for mentally retarded persons: clinical andepidemiologic observations,” J. Pedatr., 116: 544-551 [1990]; F. Proulxet al., “Randomized, controlled trial of antibiotic therapy forEscherichia coli O157:H7 enteritis,” J. Pediatr. 121: 299-303 [1992];and A. L. Carter et al., “A severe outbreak of Escherichia coliO157:H7-associated hemorrhagic colitis in a nursing home,” New Eng. J.Med., 317: 1496-1500 [1987]).

[0035] The mechanisms by which antibiotics increase the risk ofinfection or related complications might involve enhancement of toxinproduction, release of toxins from killed organisms, or alteration ofnormal competing intestinal flora allowing for pathogen overgrowth (M.A. Karmali, “Infection by Verocytotoxin-producing Escherichia coli,”Clin. Microbiol. Rev., 2: 15-38 [1989]). A further concern in the use ofantibiotics is the potential acquisition of antimicrobial resistance byE. coli O157:H7 (C. R. Dorn, “Review of foodborne outbreak ofEscherichia coli O157:H7 infection in the western United States,” JAVMA203: 1583-1587 [1993]).

[0036] In addition, by the time symptoms are serious enough to attractmedical attention, it is likely that verotoxins are already entering thesystemic circulation or will do so shortly thereafter. Althoughantimicrobials may help to prevent pathology resulting from the actionof toxin on the bowel lumen. However, by the time symptoms of HUS havedeveloped, the patient has ceased shedding organisms. Thus,antimicrobial treatment during HUS disease is of less value, and oftencontraindicated, due to the increased risk of complications associatedwith administration of antimicrobials to patients susceptible todevelopment of HUS. Importantly, there is currently no antitoxincommercially available for use in treating affected patients. What isneeded is a means to block the progression of disease, without thecomplications associated with antimicrobial treatment.

DESCRIPTION OF THE FIGURES

[0037]FIG. 1 is an SDS-PAGE of rVT1 and rVT2.

[0038]FIG. 2 shows HPLC results for rVT1 and rVT2.

[0039]FIG. 3 shows rVT1 and rVT2 toxicity in Vero cell culture.

[0040]FIG. 4 shows ELISA reactivity of RVT. and rVT2 antibodies to rVT1.

[0041]FIG. 5 shows ELISA reactivity of rVT1 and rVT2 Antibodies to rVT2.

[0042]FIG. 6 shows Western Blot reactivity of rVT1 and rVT2 antibodiesto rVTs:

[0043] Panel 6A contains preimmune IgY;

[0044] Panel 6B contains rVT1 IgY; and

[0045] Panel 6C contains rVT2 IgY.

[0046]FIG. 7 shows neutralization of rVT1 cytotoxicity in Vero cells.

[0047]FIG. 8 shows neutralization of rVT2 cytotoxicity in Vero cells.

[0048]FIG. 9 shows renal sections from E. coli O157:H7-infected micetreated with IgY:

[0049] Panel 9A shows a representative kidney section from a mousetreated with preimmune IgY;

[0050] Panel 9B shows a representative kidney sections from a mousetreated with rVT1; and

[0051] Panel 9C shows a representative kidney section from a mousetreated with rVT2 IgY.

[0052]FIG. 10 shows the fusion constructs of VT components and affinitytags.

[0053]FIG. 11 shows a representative Coomassie stained SDS-PAGE geldemonstrating purification of his-tagged VT1 B and VT2 B proteins.

[0054]FIG. 12 shows a representative Coomassie stained SDS-PAGE gel withpMa1VTIA and pMa1VT2A protein preparations.

[0055]FIG. 13 shows a representative Coomassie stained SDS-PAGE gel withpMa1VT2A and pMa1VT2A(BamHI) protein preparations.

[0056]FIG. 14 shows a representative Coomassie-stained SDS-PAGE gel withuntreated and cross-linked immunogens.

[0057]FIG. 15 shows the ELISA reactivity of VT1A IgY and VT2A IgY torVTI.

[0058]FIG. 16 shows the ELISA reactivity of VT1A IgY and VT2A IgY torVT2.

[0059]FIG. 17 shows the ELISA reactivity of VT1B IgY and VT2B IgY torVTI.

[0060]FIG. 18 shows the ELISA reactivity of VT1B IgY and VT2 B IgY torVT2.

DEFINITIONS

[0061] To facilitate understanding of the invention, a number of termsare defined be low.

[0062] As used herein, the term “neutralizing” is used in reference toantitoxins, particularly antitoxins comprising antibodies, which havethe ability to prevent the pathological actions of the toxin againstwhich the antitoxin is directed. It is * con templated that neutralizingantibodies be utilized to prevent the action of toxins, in particular E.coli verotoxins and Shiga toxin. It is further contemplated thatneutralizing antibodies be utilized to alleviate the effect(s) of toxinsin an individual, in particular E. coli verotoxins and Shiga toxin.

[0063] As used herein, the term “immunogen” refers to a substance,compound, molecule, or other moiety which stimulates the production ofan immune response. The term “antigen” refers to a substance, compound,molecule, or other moiety that is capable of reacting with products ofthe immune response. For example, verotoxin subunits may be used asilnunogens to elicit an immune response in an animal to produceantibodies directed against the subunit used as an immunogen. Thesubtuni may then be used as an antigen in an assay to detect thepresence of anti-verotoxin subunit antibodies in the serum of theimmunized animal.

[0064] As used herein, the term “overproducing” is used in reference tothe production of toxin polypeptides in a host cell, and indicates thatthe host cell is producing more of the toxin by virtue of theintroduction of nucleic acid sequences encoding the toxin polypeptidethan would be expressed by the host cell absent the introduction ofthese nucleic acid sequences. To allow ease of purification of toxinpolypeptides produced in a host cell it is preferred that the host cellexpress or overproduce the toxin polypeptide at a level greater than 1mg/liter of host cell culture.

[0065] “A host cell capable of expressing a recombinant protein as asoluble protein at a level greater than or equal to 5% of the totalsoluble cellular protein” is a host cell in which the amount of solublerecombinant protein present represents at least 5% of the total solublecellular protein. As used herein “total soluble cellular protein” refersto a clarified PEI lysate prepared as described in the Examples.Briefly, cells are harvested following induction of expression ofrecombinant protein (at a point of maximal expression). The cells areresuspended in cell resuspension buffer (CRB: 50 mM NaPO₄, 0.5 M NaCl,40 mM imidazole, pH 6.8) to create a 20% cell suspension (wet weight ofcells/volume of CRB) and clarified cell lysates are prepared asdescribed in the Examples (ie., sonication or homogenization followed bycentrifugation). The amount of purified recombinant protein (i.e., theeluted protein) is divided by the concentration of protein present inthe clarified lysate (typically 8 mg/ml when using a 20% cell suspensionas the starting material) and multiplied by 100 to determine whatpercentage of total soluble cellular protein is comprised of the solublerecombinant protein.

[0066] “A host cell capable of expressing a recombinant protein as asoluble protein at a level greater than or equal to X milligrams per 1OD of cells per liter” is a host cell that produces X milligrams ofrecombinant protein per liter of culture medium containing a density ofhost cells equal to 1 OD₆₀₀. The amount of recombinant protein presentper OD per liter is determined by quantitating the amount of recombinantprotein recovered following affinity purification. For example as shownin Ex. 8, host cells containing the pET24hisVT1 BL+construct express therecombinant VT1B protein at a level ≧1 mg rVTIB/1 OD₆₀₀/liter. Hostcells containing the pET24hisVT2BL+construct express the recombinantVT2B protein at a level ≧10 mg rVT1 B/1 OD₆₀₀/liter (See e.g., Example8).

[0067] “A host cell capable of secreting a recombinant protein into theculture supernatant at a level greater than or equal to 10 mgrecombinant protein per 1 OD of cells per liter” refers to a host cellthat secretes a recombinant protein into the culture supernatant (i.e.,the medium, such as L broth, used to grow the host cell) at a levelgreater than or equal to 10 mg recombinant protein per liter of mediumcontaining a concentration (i.e., density) of host cells equal to 1OD₆₀₀. The host cells may be grown in shaker flasks (˜1 liter culturemedium) or in fermentation tank (˜10 liters culture medium) and theamount of recombinant protein secreted into the culture supernatant maybe determined using a quantitative ELISA assay as described in Example12.

[0068] As used herein, the term “fusion protein” refers to a chimericprotein containing the protein of interest (ie., an E. coli verotoxinand/or fragments thereof) joined to an exogenous protein fragment (thefusion partner which consists of a non-toxin protein). The fusionpartner may enhance solubility of the E. coli protein as expressed in ahost cell, may provide an “affinity tag” to allow purification of therecombinant fusion protein from the host cell or culture supernatant, orboth. If desired, the fusion protein may be removed from the protein ofinterest (i.e., toxin protein or fragments thereof) prior toimmunization by a variety of enzymatic or chemical means known to theart.

[0069] As used herein the term “non-toxin protein” or “non-toxin proteinsequence” refers to that portion of a fusion protein which comprises aprotein or protein sequence which is not derived from a bacterial toxinprotein.

[0070] As used herein, the term “affinity tag” refers to such structuresas a “poly-histidine tract” or “poly-histidine tag,” or any otherstructure or compound which facilitates the purification of arecombinant fusion protein from a host cell, host cell culturesupernatant, or both. As used herein, the term “flag tag” refers toshort polypeptide marker sequence useful for recombinant proteinidentification and purification.

[0071] As used herein, the terms “poly-histidine tract” and“poly-histidine tag,” when used in reference to a fusion protein refersto the presence of two to ten histidine residues at either the amino- orcarboxy-terminus of a protein of interest. A poly-histidine tract of sixto ten residues is preferred. The poly-histidine tract is also definedfinctionally as being a number of consecutive histidine residues addedto the protein of interest which allows the affinity purification of theresulting fusion protein on a nickel-chelate or IDA column.

[0072] As used herein, the term “chimeric protein” refers to two or morecoding sequences obtained from different genes, that have been clonedtogether and that, after translation, act as a single polypeptidesequence. Chimeric proteins are also referred to as “hybrid proteins.”As used herein, the term “chimeric protein” refers to coding sequencesthat are obtained from different species of organisms, as well as codingsequences that are obtained from the same species of organisms.

[0073] As used herein, the term “protein of interest” refers to theprotein whose expression is desired within the fusion protein. In afusion protein, the protein of interest will be joined or fused withanother protein or protein domain, the fusion partner, to allow forenhanced stability of the protein of interest and/or ease ofpurification of the fusion protein.

[0074] As used herein, the term “maltose binding protein” and “MBP”refers to the maltose binding protein of E. coli. A portion of themaltose binding protein may be added to a protein of interest togenerate a fusion protein; a portion of the maltose binding protein maymerely enhance the solubility of the resulting fusion protein whenexpressed in a bacterial host. On the other hand, a portion of themaltose binding protein may allow affinity purification of the fusionprotein on an amylose resin.

[0075] As used herein, the term “purified” or “to purify” refers to theremoval of contaminants from a sample. For example, antitoxins arepurified by removal of contaminating non-immunoglobulin proteins; theyare also purified by the removal of substantially all immunoglobulinthat does not bind toxin. The removal of non-immunoglobulin proteinsand/or the removal of immunoglobulins that do not bind toxin results inan increase in the percent of toxin-reactive immunoglobulins in thesample. In another example, recombinant toxin polypeptides are expressedin bacterial host cells and the toxin polypeptides are purified by theremoval of host cell proteins; the percent of recombinant toxinpolypeptides is thereby increased in the sample.

[0076] As used herein, the term “periplasmic” refers to the space aroundthe plasma membrane, or more specifically, the space between the plasmamembrane and the cell wall of a bacterium.

[0077] The term “recombinant DNA molecule” as used herein refers to aDNA molecule which is comprised of segments of DNA joined together bymeans of molecular biological techniques.

[0078] The term “recombinant protein” or “recombinant polypeptide” asused herein refers to a protein molecule which is expressed from arecombinant DNA molecule.

[0079] The term “native protein” as used herein refers to a proteinwhich is isolated from a natural source as opposed to the production ofa protein by recombinant means.

[0080] The terms “native gene” or “native gene sequences” are used toindicate DNA sequences encoding a particular gene which contain the sameDNA sequences as found in the gene as isolated from nature. In contrast,“synthetic gene sequences” are DNA sequences which are used to replacethe naturally occurring DNA sequences when the naturally occurringsequences cause expression problems in a given host cell. For example,naturally-occurring DNA sequences encoding codons which are rarely usedin a host cell may be replaced (e.g., by site-directed mutagenesis) suchthat the synthetic DNA sequence represents a more frequently used codon.The native DNA sequence and the synthetic DNA sequence will preferablyencode the same amino acid sequence.

[0081] As used herein the term “portion” when in reference to a protein(as in “a portion of a given protein”) refers to fragments of thatprotein. The fragments may range in size from four amino acid residuesto the entire amino acid sequence minus one amino acid.

[0082] As used herein “soluble” when in reference to a protein producedby recombinant DNA technology in a host cell, is a protein which existsin solution in the cytoplasm of the host cell; if the protein contains asignal sequence, the soluble protein is exported to the periplasmicspace in bacterial hosts and is secreted into the culture medium ofeukaryotic cells capable of secretion or by bacterial hosts possessingthe appropriate genes. In contrast, an insoluble protein is one whichexists in denatured form inside cytoplasmic granules (ie., inclusionbodies) in the host cell. High level expression (i.e., greater than 1 mgrecombinant protein/liter of bacterial culture) of recombinant proteinsoften results in the expressed protein being found in inclusion bodiesin the bacterial host cells. A soluble protein is a protein which is notfound in an inclusion body inside the host cell or is found both in thecytoplasm and in inclusion bodies and in this case the protein may bepresent at high or low levels in the cytoplasm.

[0083] A distinction is drawn between a soluble protein (i.e., a proteinwhich when expressed in a host cell is produced in a soluble form) and a“solubilized” protein. An insoluble recombinant protein found inside aninclusion body may be solubilized (ie., rendered into a soluble form) bytreating purified inclusion bodies with denaturants such as guanidinehydrochloride, urea or sodium dodecyl sulfate (SDS). These denaturantsmust then be removed from the solubilized protein preparation to allowthe recovered protein to renature (refold). Not all proteins will refoldinto an active conformation after solubilization in a denaturant andremoval of the denaturant. Many proteins precipitate upon removal of thedenaturant. SDS may be used to solubilize inclusion bodies and willmaintain the proteins in solution at low concentration. However,dialysis will not always remove all of the SDS (SDS can form micelleswhich do not dialyze out); therefore, SDS-solubilized inclusion bodyprotein is soluble but not refolded.

[0084] A distinction is drawn between proteins which are soluble ( i.e.,dissolved) in a solution devoid of significant amounts of ionicdetergents (e.g., SDS) or denaturants (e.g., urea, guanidinehydrochloride) and proteins which exist as a suspension of insolubleprotein molecules dispersed within the solution. A soluble protein willnot be removed from a solution containing the protein by centrifugationusing conditions sufficient to remove bacteria present in a liquidmedium (i.e., centrifugation at 12,000×g for 4-5 minutes). For example,to test whether two proteins, protein A and protein B, are soluble insolution, the two proteins are placed into a solution selected from thegroup consisting of PBS-NaCl (PBS containing 0.5 M NaCI), PBS-NaClcontaining 0.2% Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBScontaining 2 mM CaCl₂), PBS-C containing either 0.1 or 0.5 % Tween 20,PBS-C containing either 0.1 or 0.5% NP-40, PBS-C containing either 0.1or 0.5% Triton X-100, PBS-C containing 0.1% sodium deoxycholate. Themixture containing proteins A and B is then centrifuged at 5000×g for 5minutes. The supernatant and pellet formed by centrifugation are thenassayed for the presence of protein A and B. If protein A is found inthe supernatant and not in the pellet [except for minor amounts (i.e.,less than 10%) as a result of trapping], protein is said to be solublein the solution tested. If the majority of protein B is found in thepellet (i.e., greater than 90%), then protein B is Isaid to exist as asuspension in the solution tested.

[0085] As used herein, the term “reporter reagent” or “reportermolecule” is used in reference to compounds which are capable ofdetecting the presence of antibody bound to antigen. For example, areporter reagent may be a colorimetric substance which is attached to anenzymatic substrate. Upon binding of antibody and antigen, the enzymeacts on its substrate and causes the production of a color. Otherreporter reagents include, but are not limited to fluorogenic andradioactive compounds or molecules.

[0086] As used herein the term “signal” is used in reference to theproduction of a sign that a reaction has occurred, for example, bindingof antibody to antigen. It is contemplated that signals in the form ofradioactivity, fluorogenic reactions, and enzymatic reactions will beused with the present invention. The signal may be assessedquantitatively as well as qualitatively.

[0087] As used herein, the term “therapeutic amount” refers to thatamount of antitoxin required to neutralize the pathologic effects of E.coli toxin in a subject.

[0088] As used herein, the term “acute intoxication” is used inreference to cases of E. coli infection in which the patient iscurrently suffering from the effects of toxin (e.g., E. coli verotoxinsor enterotoxins). Signs and symptoms of intoxication with the toxin maybe immediately apparent. Or, the determination of intoxication mayrequire additional testing, such as detection of toxin present in thepatient's fecal material.

[0089] As used herein, the term “at risk” is used in references toindividuals who have been exposed to E. coli and may suffer the symptomsassociated with infection or disease with these organisms, especiallydue to the effects of verotoxins.

[0090] The term “pyrogen” as used herein refers to a fever-producingsubstance. Pyrogens may be endogenous to the host (e.g., prostaglandins)or may be exogenous compounds (e.g., bacterial endo- and exotoxins,non-bacterial compounds such as antigens and certain steroid compounds,etc.). The presence of pyrogen in a pharmaceutical solution may bedetected using the U.S. Pharmacopeia (USP) rabbit fever test (UnitedStates Pharmacopeia, Vol. XXII (1990) United States PharmacopeialConvention, Rockville, Md., p. 151).

[0091] The term “endotoxin” as used herein refers to the high molecularweight complexes associated with the outer membrane of gram-negativebacteria. Unpurified endotoxin contains lipids, proteins andcarbohydrates. Highly purified endotoxin does not contain protein and isreferred to as lipopolysaccharide (LPS). Because unpurified endotoxin isof concern in the production of pharmaceutical compounds (e.g., proteinsproduced in E. coli using recombinant DNA technology), the termendotoxin as used herein refers to unpurified endotoxin. Bacterialendotoxin is a well known pyrogen.

[0092] As used herein, the term “endotoxin-free” when used in referenceto a composition to be administered parenterally (with the exception ofintrathecal administration) to a host means that the dose to bedelivered contains less than 5 EU/kg body weight (FDA Guidelines forParenteral Drugs [December 1987]). Assuming a weight of 70 kg for anadult human, the dose must contain less than 350 EU to meet FDAGuidelines for parenteral administration. Endotoxin levels are measuredherein using the Limulus Amebocyte Lysate (LAL) test (Limulus AmebocyteLysate Pyrochrome™, Associates of Cape Cod, Inc. Woods Hole, Mass.). Tomeasure endotoxin levels in preparations of recombinant proteins, 0.5 mlof a solution comprising 0.5 mg of purified recombinant protein in 50 mMNaPO₄, pH 7.0, 0.3 M NaCl and 10% glycerol is used in the LAL assayaccording to the manufacturer's instructions for the endpointchromogenic without diazo-coupling method [the specific components ofthe buffer containing recombinant protein to be analyzed in the LAL testare not important; any buffer having a neutral pH may be employed.Compositions containing less than or equal to than 250 endotoxin units(EU)/mg of purified recombinant protein are herein defined as“substantially endotoxin-free.” Preferably the composition contains lessthan or equal to 100, and most preferably less than or equal to 60,(EU)/mg of purified recombinant protein. Typically, administration ofbacterial toxins or toxoids to adult humans for the purpose ofvaccination involves doses of about 10-500 μg protein/dose. Therefore,administration of 10-500 μg of a purified recombinant protein to a 70 kghuman, wherein said Th. purified recombinant protein preparationcontains 60 EU/mg protein, results in the introduction of only 0.6 to 30EU (i.e., 0.2 to 8.6% of the maximum allowable endotoxin burden perparenteral dose). Administration of 10-500 μg of a purified recombinantprotein to a 70 kg human, wherein said purified recombinant proteinpreparation contains 250 EU/mg protein, results in the introduction ofonly 2.5 to 125 EU (i.e., 0.7 to 36% of the maximum allowable endotoxinburden per parenteral dose).

[0093] The LAL test is accepted by the U.S. FDA as a means of detectingbacterial endotoxins (21 C.F.R. §§ 660.100 -105). Studies have shownthat the LAL test is equivalent or superior to the USP rabbit pyrogentest for the detection of endotoxin and thus the LAL test can be used asa surrogate for pyrogenicity studies in animals (F. C. Perason,Pyrogens: endotoxins, LAL Testing and Depyrogenation, Marcel Dekker, NewYork [1985], pp.150-155). The FDA Bureau of Biologics accepts the LALassay in place of the USP rabbit pyrogen test so long as the LAL assayutilized is shown to be as sensitive as, or more sensitive as the rabbittest (Fed. Reg., 38, 26130 (1980]).

[0094] The term “monovalent” when used in reference to a verotoxinvaccine refers to a vaccine which is capable of provoking an immuneresponse in a host animal directed against a single type of verotoxin.For example, if immunization of a host with E. coli VT1 toxin vaccineinduces antibodies in the immunized host which protect against achallenge with VT1, but not against challenge with other toxins (e.g.,VT2), then the VT1 vaccine is said to be monovalent. In contrast, a“multivalent” vaccine provokes an immune response in a host animaldirected against more than one verotoxin. For example, if immunizationof a host with a vaccine comprising VT1 and VT2 verotoxins induces theproduction of antibodies which protect (ie., “protective antibody”) thehost against a challenge with both VT1 and VT2, the vaccine is said tobe multivalent (in particular, this hypothetical vaccine is bivalent).It is intended that multivalent vaccines of the present inventionencompass numerous embodiments. For example, it is also contemplatedthat recombinant E. coli verotoxin proteins be used in conjunction witheither native toxins or toxoids from other organisms as antigens in amultivalent vaccine preparation. It is further contemplated thatmultivalent vaccines of the present invention will stimulate an immuneresponse against various E. coli serotypes, including, but not limitedto E. coli O157:H7, O216:H11, O113:H21, 091 :H21, and 0111 :NM, inhumans and/or other animals.

[0095] As used herein the term “immunogenically-effective amount” refersto that amount of an immunogen required to invoke the production ofprotective levels of antibodies in a host upon vaccination.

[0096] The term “protective level”, when used in reference to the levelof antibodies induced upon immunization of the host with an immunogenwhich comprises a bacterial toxin, means a level of circulatingantibodies sufficient to protect the host from challenge with a lethalor an detrimental dose of the toxin.

[0097] As used herein the terms “protein” and “polypeptide” refer tocompounds comprising amino acids joined via peptide bonds and are usedinterchangeably.

[0098] The terms “toxin” when used in reference to toxins produced bymembers (i.e., species and strains) of the genera Escherichia andShigella are used interchangeably and refer to verotoxins, Shigatoxin,or Shiga-like toxins

[0099] The term “receptor-binding domain” when used in reference toverotoxin refers to the area of the B subunit presumed to be responsiblefor the binding of the holotoxin to the receptor on the targeteukaryotic cell. The receptor for VT1 and VT2 is a globotriaosylceramide containing a galactose α-(1-4)- galactose-β-(1-4) glucoseceramide (Gb3). The present invention contemplates fusion proteinscomprising the receptor-binding domain of verotoxins (e.g., VT1 and VT2)from E. coli, including the variants found among different strainswithin a given serotype, in particular E. coli O157:H7. Fusion proteinscontaining an analogous region from a strain other than the prototypestrain are encompassed by the present invention.

[0100] Fusion proteins comprising the receptor binding domain (ie., theB subunit) of botulinal toxins may include amino acid residues locatedbeyond the termini of the domains defined above.

SUMMARY OF THE INVENTION

[0101] The present invention relates to antitoxin therapy for humans andother animals. Antitoxins which neutralize the pathologic effects of E.coli toxins are generated by immunization of avian hosts withrecombinant toxin fragments. In one embodiment, the present inventioncontemplates a method of treatment administering at least one antitoxindirected against at least a portion of an Escherichia coli verotoxin inan aqueous solution in therapeutic amount that is administrable to anintoxicated subject. It is contemplated that the intoxicated subjectwill be either an adult or a child.

[0102] In a preferred embodiment, the E. coli verotoxin is recombinant.In one embodiment, the antitoxin is an avian antitoxin. In analternative embodiment, the recombinant E. coli verotoxin is a fusionprotein comprising a non-verotoxin protein sequence and a portion of theEscherichia coli verotoxin VT1 sequence. In one embodiment of the E.coli fusion protein, the fusion protein comprises a non-verotoxinprotein sequence and a portion of the Escherichia coli verotoxin VT2sequence.

[0103] Various routes of administration, are contemplated for providingthe E. coli antitoxin(s) to an affected individual, including but notlimited to, parenteral as well as oral routes of administration. In aparticularly preferred embodiment, the route of administration isparenteral.

[0104] The present invention also includes the embodiment of a method ofprophylactic treatment in which an antitoxin directed against at leastone E. coli verotoxin in an aqueous solution in therapeutic amount thatis parenterally administrable, and is administered to at least onesubject at risk of diarrheal disease. It one embodiment, the antitoxinis parenterally administered.

[0105] In one embodiment, the subject is at risk of developingextra-intestinal complications of E. coli infections, including but notlimited to, hemolytic uremic syndrome, thrombotic thrombocytopenicpurpura, etc.

[0106] The present invention also includes the embodiment of acomposition which comprises neutralizing antitoxin directed against atleast one E. coli verotoxin in an aqueous solution in therapeuticamounts. In one particularly preferred embodiment, the E. coli verotoxinis a recombinant toxin. In an alternative embodiment, the recombinant E.coli verotoxin is a fusion protein comprising a non-verotoxin proteinsequence and a portion of the E. coli verotoxin VT1 sequence. In anotherembodiment, the recombinant E. coli verotoxin is a fusion proteincomprising a non-verotoxin protein sequence and a portion of the E. coliverotoxin VT2 sequence. In yet another embodiment, the composition ofthe antitoxin is directed against a portion of at least one Escherichiacoli verotoxin. In one embodiment, the portion of Escherichia coli isselected from the group consisting of subunit A and subunit B of VT1. Inan alternative embodiment, the portion of Escherichia coli is selectedfrom the group consisting of subunit A and subunit B of VT2. Indeed, theinvention contemplates an antitoxin that is directed against a portionof at least one Escherichia coli verotoxin. In one embodiment, theantitoxin is an avian antitoxin.

[0107] The present invention also comprises a method of treatment ofenteric bacterial infections comprising administering an avian antitoxindirected against at least one verotoxin produced by E. coli in anaqueous solution in therapeutic amount, to at least one infectedsubject. In one preferred embodiment, the avian antitoxin isadministered parenterally.

[0108] In another embodiment, the E. coli is selected from the groupconsisting of Escherichia coli serotypes O157:H7, O1:NM; O2:H5; 02:H7;04:NM; O4:H10; O5:NM; O5:H16; O6:H1; O18:NM; O18:H7; 025:NM; O26:NM;O26:H11; O26:H32; O38:H21; O39:H4; O45:H2; O50:H7; 055:H7; 055:H10;O82:H8; O84:H2; O91:NM; O91:H21; O103:H2; O111:NM; O111:H8; O111:H30;O111:H34; O113:H7; O113:H21; O114:H48; O115:H10; O117:H4; O118:H12;O118:H30; O121:NM; O121:H19; O125:NM; O125:H8; O126:NM; O126:H8;O128:NM; O128:H2; O128:H8; O128:H12; O128:H25; O145:NM; O125:H25;O146:H21; O153:H25; O157:NM; O163:H19; O165:NM; O165:19; and O165:H25.In one embodiment, the antitoxin comprises antitoxin directed against atleast one Escherichia coli verotoxin. In another embodiment, theantitoxin is cross-reacts with at least one Escherichia coli verotoxin.In yet another embodiment, the antitoxin is reactive against toxinsproduced by members of the genus Shigella, including S. dysenteriae.

[0109] The present invention also contemplates uses for the toxinfragments in vaccines and diagnostic assays. The fragments may be usedseparately as purified, soluble antigens or, alternatively, in mixturesor “cocktails.” The present invention thus comprises a method fordetecting Escherichia coli verotoxin in a sample in which a sample,anantitoxin raised against Escherichia coli verotoxin, and a reporterreagent capable of binding the antitoxin are provided. The antitoxin isadded to the sample, so that the antitoxin binds to the E. coliverotoxin in the sample. In one embodiment, the antitoxin is an avianantitoxin. In an alternative embodiment, the method further comprisesthe steps of washing unbound antitoxin from the sample, adding at leastone reporter reagent to the sample, so that the reporter reagent bindsto any antitoxin that is bound, washing the unbound reporter reagentfrom the sample and detecting the reporter reagent bound to theantitoxin bound to the Escherichia coli verotoxin, so that the verotoxinis detected. In one embodiment, the detecting is accomplished throughany means, such as enzyme immunoassay, radioimmunoassay, fluorescenceimmunoassay, flocculation, particle agglutination, and in situchromogenic assay. In one preferred embodiment, the sample is abiological sample. In an alternative preferred embodiment, the sample isan environmental sample.

[0110] The present invention also provides a recombinant expressionvector, in which the vector encodes an affinity tag and proteincomprising at least a portion of bacterial toxin selected from the groupconsisting of Escherichia coli type 1 verotoxin and Escherichia colitype 2 verotoxin. In preferred embodiments, the vector comprises nucleicacid encoding at least a portion of an amino acid sequence selected fromthe group consisting of SEQ ID NOS:3, 8, 21, 23, 25, 27, 47 and 49. Inparticularly preferred embodiments, the affinity tag comprises apolyhistidine tract or the maltose binding protein.

[0111] In one embodiment, the recombinant expression vector contains aportion of Escherichia coli type 1 verotoxin selected from the groupconsisting of Escherichia coli type 1 verotoxin subunit A, Escherichiacoli type 1 verotoxin subunit B, Escherichia coli type 2 verotoxinsubunit A, and Escherichia coli type 2 verotoxin subunit B. In preferredembodiments, the affinity tag is selected from the group consisting of apolyhistidine tract and maltose binding protein.

[0112] In yet another embodiment, the present invention provides a hostcell capable of expressing a recombinant verotoxin protein as a solubleprotein at a level of at least 1 milligram per 1 OD of the host cellsper liter. In an alternative embodiment, the recombinant verotoxinprotein is expressed as a soluble protein at a level of at least 10milligrams per 1 OD of the host cells per liter.

[0113] In an alternative embodiment, the present invention provides ahost cell containing a recombinant expression vector, the vectorencoding an affinity tag and protein comprising at least a portion ofbacterial toxin, the toxin selected from the group consisting ofEscherichia coli type 1 verotoxin, Escherichia coli type 2 verotoxin,and Shiga toxin. In preferred embodiments, the host cell contains anexpression vector selected from the group consisting of pET24hisVT2BL+,pET24hisVTIBL+, and pET23hisVT2AL−.

[0114] In yet another embodiment, the present invention provides a hostcell expressing toxin portion that is selected from the group consistingof Escherichia coli type 1 verotoxin subunit A, Escherichia coli type 1verotoxin subunit B, Escherichia coli type 2 verotoxin subunit A, andEscherichia coli type 2 verotoxin subunit B.

[0115] In preferred embodiments, the host cell is a bacterial cell. Inparticularly preferred embodiments, the host cell is an Escherichia colicell, in another embodiment, the host cell is a Shigella cell.

[0116] In alternatively preferred embodiments, the host cell of thepresent invention is an eukaryotic cell. In preferred embodiments, thehost cell is an insect, yeast, or mammalian cell.

[0117] The present invention also provides a host cell containing arecombinant expression vector, in which the vector encodes a fusionprotein comprising a non-toxin protein sequence and at least a portionof a bacterial toxin, wherein the toxin selected from the groupconsisting of Escherichia coli type 1 verotoxin, Escherichia coli type 2verotoxin, and Shiga toxin.

[0118] In alternative embodiments, the host cell contains Escherichiacoli verotoxin portion is selected from the group consisting ofEscherichia coli type 1 verotoxin subunit A, Escherichia coli type 1verotoxin subunit B, Escherichia coli type 2 verotoxin subunit A, andEscherichia coli type 2 verotoxin subunit B. In particularly preferredembodiments, the non-toxin protein sequence is selected from the groupcomprising a poly-histidine tract and the maltose binding protein.

[0119] The present invention also provides methods of generatingneutralizing antibody directed against Escherichia coli verotoxincomprising: providing in any order: an antigen comprising a fusionprotein comprising a non-toxin protein sequence and at least a portionof a Escherichia coli verotoxin, the toxin selected from the groupconsisting of type 1 toxin and type 2 toxin, a host; and immunizing thehost with the antigen so as to generate a neutralizing antibody.

[0120] In preferred embodiments of the method, the antigen furthercomprises a fusion protein comprising a non-toxin protein sequence andat least a portion of Escherichia coli verotoxin selected from the groupcomprising Escherichia coli type 1 verotoxin and Escherichia coli type 2verotoxin. In alternatively preferred embodiments, the antigen iscross-linked. In particularly preferred embodiments, the non-toxinprotein sequence comprises a poly-histidine tract or the maltose bindingprotein. In preferred embodiments of the method, the host is a chicken,mammal (including humans).

[0121] In yet another preferred embodiment, the methods further compristhe step of collecting antibodies from the host. In yet anotheralternative embodiment, the methods further comprise the step ofpurifying the antibodies to provide an antibody preparation. Inparticularly preferred embodiments of the method, the purifyingcomprises affinity purification. In alternatively preferred embodiments,the purified antibody preparation contains 0.2 to 1% specific antibody.

[0122] The present invention also provides the antibody preparedaccording to the methods described above. In particular embodiments, theantibody raised according to the methods wherein the produced antibodyis an avian antibody. In alternatively preferred embodiments, theantibody raised according to the methods, wherein the antibody isprotective.

[0123] The present invention also provides methods of treatmentcomprising: providing: neutralizing antitoxin directed against at leasta portion of an Escherichia coli recombinant verotoxin in an aqueoussolution in therapeutic amount that is administrable, and an intoxicatedsubject; and administering the antitoxin to the subject. In preferredembodiments, the antitoxin is an avian antitoxin, while in alternativepreferred embodiments, the antitoxin is a mammalian antitoxin.

[0124] In alternative embodiments of the methods, the recombinantEscherichia coli verotoxin is a fusion protein comprising anon-verotoxin protein sequence and a portion of Escherichia coliverotoxin VT1 sequence. In alternatively preferred embodiments, therecombinant Escherichia coli verotoxin is a fusion protein comprising anon-verotoxin protein sequence and a portion of Escherichia coliverotoxin VT sequence selected from the group comprising the VT1 subunitA sequence, the VT1 subunit B sequence, VT2 subunit A sequence, and VT2subunit B sequence. In alternative preferred embodiments, therecombinant Escherichia coli verotoxin is a fusion protein iscross-linked.

[0125] It is contemplated that the subject of the methods be either anadult or a child. It is further contemplated that the administering beaccomplished by various methods, including but not limited toparenteral.or oral.

[0126] The present invention also provides methods of prophylactictreatment comprising: providing: a neutralizing antitoxin directedagainst at least one Escherichia coli recombinant verotoxin in anaqueous solution in therapeutic amount that is parenterallyadministrable, and at least one subject is at risk of diarrheal disease;and parenterally administering the antitoxin to the subject.

[0127] In preferred embodiments of the methods, the antitoxin directedagainst Escherichia coli recombinant verotoxin is directed againstEscherichia coli verotoxin type 1, or type 2. In particularly preferredembodiments, the antitoxin is directed against Escherichia colirecombinant verotoxin is directed against Escherichia coli verotoxintype 1 subunit B or verotoxin type 2 subunit B.

[0128] In alternatively preferred embodiments, the subject is at risk ofdeveloping extra-intestinal complications of Escherichia coli infection.It is also contemplated that the subject be at risk of or experiencingextra-intestinal complication is hemolytic uremic syndrome.

[0129] The present invention also provides vaccines comprising a fusionprotein, the fusion protein comprising a non-toxin protein sequence andat least a portion of a bacterial toxin, the verotoxin selected from thegroup consisting of Escherichia coli type 1 verotoxin, Escherichia colitype 2 verotoxin, and Shiga toxin.

[0130] In preferred embodiments, the vaccine further comprises a fusionprotein comprising a non-toxin protein sequence and at least a portionof Escherichia coli verotoxin type 1 verotoxin. In alternative preferredembodiments, the vaccine further comprises a fusion protein comprising anon-toxin protein sequence and at least a portion of Escherichia coliverotoxin type 2 verotoxin. In yet other alternative embodiments, thevaccine comprises non-toxin protein sequence selected from the groupconsisting of poly-histidine tract and maltose binding protein. In yetother alternative embodiments, vaccine substantially endotoxin-free. Infurther alternative embodiments, the bacterial toxin is cross-linked.

DESCRIPTION OF THE INVENTION

[0131] The present invention contemplates preventing or treating humansand other animals intoxicated with at least one bacterial toxin. It iscontemplated that administration of antitoxin will be used to treatpatients effected by or at risk of symptoms due to the action ofbacterial toxins. It is also contemplated that the antitoxin will beused in a diagnostic assay to detect the presence of toxins in samples.The present invention further provides methods for preparation andutilization of immunogens and antigens. In preferred embodiments, thesepreparations are obtained using recombinant methodologies. Theimmunogens, organisms, toxins and individual steps of the presentinvention are described separately below.

[0132] I. Production of Immunogens/Antigens

[0133] In preferred embodiments, the present invention provides methodsfor the over-expression of VT1 and VT2 subunits. Cloning of the VT1 andVT2 gene clusters on high copy number plasmids (e.g., pUC19 or pBS;Stratagene) has been used to over-express the toxins in E. coli . Insome instances, periplasmic extracts were utilized for biochemicalpurification of the toxins. However, this approach of coordinateexpression of toxin subunits has disadvantages, such as the inherenttoxicity of the toxins on the cell lines, which necessitates the use ofspecialized facilities for growth of organisms and purification of thetoxins.

[0134] A and B chains of VT1 and VT2 have also individually expressed inE. coli , utilizing recombinant DNA methodologies. However, the presentinvention provides methods for the expression of toxin subunits fromhigh copy number plasmids or expression vectors, periplasmic secretion,and ready purification of folded protein. The location of expression inthe periplasmic space is very desirable, as the A and B subunits containdisulfide bonds that cannot be formed intracellularly due to thereducing environment. In addition, in order to be conformationallycorrect, the B subunit must be able to associate into pentamers.

[0135] In the present invention, the pET vector derived verotoxinexpression constructs were transformed into E. coli strain B121(DE3). Alisting of several plasmid constructs can be found in Table 10, below.Each plasmid containing a his-tagged subunit was tested for its effecton the viability of the host strain (i.e., BL21[DE3]). In general,constructs that did not tightly repress the expression of therecombinant subunit through the use of the T71ac promoter/Laclq genewere lethal to the strain. Plasmids that were not stable in BL21(DE3)were also tested in strains harboring plysS or plysE. These plasmidscontain the gene for T7 lysozyme, a natural inhibitor of T7 RNApolymerase. Co-expression of the lysS or lysE gene typically preventedcell death caused by the subunit.

[0136] In the present invention, protein expression in culturesutilizing recombinant plasmids in the B121 (DE3) derived E. coli strainswas induced by addition of IPTG. For optimal protein expression,cultures were grown at 30-32° C. overnight, induced when the celldensity reached >2 OD₆₀₀. Induced protein was then allowed to accumulatefor 2-4 hrs after induction. Induction at lower OD₆₀₀ readings (e.g.,0.50-1.0) resulted in accumulation of lower overall levels of verotoxinsubunits, since induction of subunit expression halted E. coli cellgrowth and final cell pellets were therefore dramatically smaller. Inthe case of VT1 B and VT2 B-expressing constructs, the verotoxinsubunits were insoluble if grown and induced at 37° C.

[0137] Although high level expression of the VT1 B subunit has beenattainable in either E. coli or V. cholerae, prior to the presentinvention, biochemical purification procedures have resulted insuboptimal recovery of the subunit from periplasmic extracts. Thepresent invention addresses these problems known in the art, in order torecover relatively high levels of expressed subunit from small andlarge-scale cultures.

[0138] In regards to VT2, very low level expression of the A subunithave been previously reported, while expression levels of solubleaffinity purifiable GST-B subunit fusion were reported to be on theorder of 1 mg/liter. Since this expression is intracellular, thepurified proteins produced are unlikely to be conformationally correct.

[0139] Several expression constructs to overexpress the VT2 B subunitseither periplasmically or cytoplasmically have been reported (Acheson etal, Infect. Immun. 63, 301-308 [1995]). In these constructs the VT2 Bsubunit was overexpressed in E. coli under the control of the T7 or tacpromoter. However, the VT2 B subunits expressed utilizing these systemsappeared to form unstable multimers, indicating that coassembly with theA subunit is necessary to form stable pentamers. Although high levelexpression was reported, only low yields of purified protein wererecovered (i.e., 1 mg from a 10 liter fermentation).

[0140] The present invention provides methods for the periplasmicexpression of individual verotoxin subunits in E. coli that areapparently conformationally correct, and can be assembled in vivo withpurified A subunit holotoxin to produce holotoxin that isconformationally and functionally identical to purified nativeholotoxin. Intracellular expression of subunits, while potentiallyyielding higher levels of expression, might require refolding strategiesto obtain native conformation. The development of large scalepurification methods of the present invention, such as those in someembodiments, that utilize the incorporation of an affinity tag tofacilitate single step affinity purification of subunits fromperiplasmic extracts, greatly enhances and simplifies the priorpurification schemes based on biochemical (Donohue-Rolfe, [1991]),immunoaffinity chromatography (Donohue-Rolfe et al., supra [1984]), orligand binding (Donohue-Rolfe et al., supra [1989]) strategies.

[0141] Affinity tagging VT-1 and VT-2. To maintain protein conformationof the B subunits, the incorporated affinity tag must not interfere withsubunit folding or pentamer formation. During the development of thepresent invention, molecular dissections of the VT1 B and VT2 Bsubunits, as well as X-ray crystallography data, indicated thatC-terminally tagged B chains may be functionally and conformationallyunaltered. The crystal structure of VT1 B chain pentamers (Stein et al.,supra [1992]) and Shiga holotoxin (Fraser et al., supra [1994]) havebeen solved. Based on these structures and mutagenesis of the B chain ofVT1 and VT2, purification of recombinant B chains using a C-terminalepitope tag was accomplished.

[0142] In addition, neutralizing epitopes are present at both the N- andC terminal regions of Shiga toxin B, since polyclonal antibodies raisedagainst peptides from these regions (i.e, aa5-18, 13-26, 7-26 from the Nterminal, and aa54-67 and 57-67) show partial neutralization of Shigatoxin (Harari,[1990]; Harari, [1988]). It was contemplated thatalteration of the N or C terminal by addition of an affinity tag mayalter these epitopes. Indeed, it has been reported that deletion of thelast 5 amino acids of Shiga toxin B (Jackson et al., supra [1990]) or 4amino acids of VT2 B (Perera et al., supra) eliminate toxin activity,while deletion of the last 2 aa of VT2 B subunit reduced cytotoxicity(Perera et al., supra). However, the addition of an 18 or 21 aaextension to the C terminus of the VT2 B chain was presumablyconformationally correct and facilitated pentamer and holotoxin assemblysince these proteins assembled cytotoxic holotoxin (Perera et al.,supra). These C terminal extensions did not alter binding of amonoclonal antibody reactive to the C terminal of the B chain (Perera etal., supra). These results indicated that addition of a small affinitytag to the C terminal of the VT2, and perhaps VT1, B chain may not alterconformation or pentamer assembly.

[0143] Although a large variety of expression systems have been designedto facilitate expression of affinity tagged fusion proteins in E. coli ,most systems utilize relatively large affinity tags, such as maltosebinding protein, protein A, B-galactosidase, Glutathione S-transferaseor thioredoxin, in which the affinity tag represents 12->100 kd in size.In view of the fact that the VT1 and VT2 B chains are only approximately8 kd in size, utilization of such a large affinity tag was viewed duringthe development of the present invention, as being likely to compromisethe activity of the subunit. Instead, the present invention provides asmall (<1 kd) 6X histidine sequence (i.e., His-His-His-His-His-His) asan affinity tag as. This sequence was found to bind to immobilized metalions at an affinity as high as 1013 (for Ni ions). This allows thesimple one step purification of fusion proteins containing this tag fromcell lysates by immobilized metal affinity chromatography (IMAC) of thepresent invention. Such purifications are scaleable to facilitatepurification of gram quantities of protein, and, due to the absence of apolyhistidine tag on E. coli proteins, provides yields of highly pureprotein preparations. Furthermore, the poly-his tag is non-immunogenic,such that most antibodies raised against poly-his containing proteinswill be specific to the fusion partner. In most cases this alleviatesthe need to cleave the fusion peptide from this tag before immunization.

[0144] However, it is contemplated that for expression of VT1 A and VT2A chains, a number of N or C terminal affinity tags may be utilized,since this subunit does not form multimers, and downstream assembly of Aand B chains to form holotoxin will not be performed.

[0145] The present invention provides methods for the expression andpurification of large quantities (e.g., 40 mg/l) of the VT2 B subunit.However, it was observed that due to the toxicity of the VT2 B subunit,strict uninduced promoter control is necessary to permit cell viability.In one embodiment, this is accomplished by co-expression with the plysEplasmid (for the pET23hisVT2BL+plasmid), or the presence of the laclqgene and the T7 lac promoter (for the pET24hisVT2BL+and L− plasmids).

[0146] Due to the need for disulfide bond formation and pentamerassembly, the vectors that allow periplasmic secretion of the protein(L+) are preferable, since intracellular E. coli is a reducingenvironment. Due to scale-up and plasmid stability concerns, inpreferred embodiments, the pET24 construct is preferable to the pET 23construct.

[0147] The present invention also provides methods for the expressionand purification of moderate quantities (5 mg/1) of the VT1 B subunit.The VT1 B subunit is less toxic than the VT2 B subunit, allowing lessstringent control of uninduced verotoxin expression. Nonetheless, due tothe need for disulfide bond formation and pentamer assembly, inpreferred embodiments, the vectors that allow periplasmic secretion ofthe protein (L+) are preferable, since intracellular E. coli is areducing environment. Due to scale-up and plasmid stability concerns,the pET24 construct is preferable to the pET 23 construct.

[0148] Due to the poor recovery of his-tagged VT1 A and VT2 A protein,expression of MBP fused VT1 A and VT2 A subunits was undertaken.Cultures of pMa1VT1 A, pMa1VT2A and pMa1VT2A (BamHI) were grown in theB121 plysS strain, induced with IPTG, and the soluble protein fractionswere isolated. Soluble extracts were bound to an amylose resin column,washed and eluted with maltose. In these embodiments, protein yieldswere 22 mg/l (pMa1VTIA), 13.5 mg/l (pMa1VT2A) or 12.5 mg/l(pMaIVT2A[BamHI]).

[0149] The identity of the predicted full length VT1 A and VT2 Aproteins was confirmed by Western blot analysis. Verotoxin protein wasidentified utilizing a chicken anti-VT1 holotoxin antiserum. Thisanalysis confirmed that the full length proteins detected by Coomassiegel analysis were immunoreactive with the anti-VT1 antibody preparation.The reactivity of the VT2 A protein with the VT1 antiserum waspredicted, since in early experiments undertaken during the developmentof the present invention, the VT1 antiserum was demonstrated tocross-react with the VT2 protein.

[0150] From Coomassie gel staining it was estimated that 50% of thepMa1VTIA elution and 10% of the pMa1VT2A(BamiHI) elution is full lengthfusion protein. This corresponds to 11 mg/liter (VT1 A) or 1.25 mg/l(VT2 A) yields of full length verotoxin subunit using these expressionsystems and embodiments.

[0151] II. Antibodies Directed Against E. coli and Associated Toxins

[0152] A preferred embodiment of the method of the present invention isdirected toward obtaining antibodies against various E. coli serotypes,their toxins, enzymes or other metabolic by-products, cell wallcomponents, or synthetic or recombinant versions of any of thesecompounds. It is contemplated that these antibodies will be produced byimmunization of humans or other animals. It is not intended that thepresent invention be limited to any particular toxin or any species oforganism. In one embodiment, toxins from all E. coli serotypes arecontemplated as immunogens. Examples of these toxins include theverotoxins VT1 and VT2.

[0153] A. Antibodies Against the A and B Subunits

[0154] As the N-terminal of both the A and B subunits are exposed, itwas contemplated that these structures may be targets for neutralizingimmune responses. Boyd et al. (Boyd et al., Infect. Immun., 59:750-757[1991]) reported that polyclonal antibodies raised against the B chainof VT-l/Shiga toxin neutralized cytotoxicity or lethality in animalmodels. However, success has not been consistently achieved. Whilepolyclonal antibodies against synthetic peptides of intervals 28-40 ofthe B subunit of VT1 neutralized cytotoxicity, while polyclonalantibodies directed against intervals 1-25 and 53-69 recognized onlydenatured forms of the B chain and failed to neutralize cytotoxicity(Boyd et al., Infect. Imrnun. 59, 750-757 [1991]). In general,neutralization of toxicity and in vivo protection have been observedwith antibodies directed against both the A and B chains of shiga toxinor VT1 . However, most analysis has implicated the B chain as the besttarget for the generation of neutralizing antibodies and, in general,neutralizing titers are highest when conformationally correct B subunitis used as immunogen, rather than linear peptides (Boyd et al, 1991,Infect. Immun. 59, 750-757). Although an understanding of precisemechanisms is not necessary for the successful practice of the presentinvention, it is contemplated that in order to obtain maximallyneutralizing antibodies, raising and testing (e.g., neutralization)polyclonal antibodies specific to both the A and B chain, relative toantibodies raised against holotoxin. Furthermore, in order to generate ahigh titer of neutralizing antibodies, the recombinant A and B chainsshould be conformationally correct.

[0155] In one embodiment of the present invention, laying Leghorn henswere immunized with the recombinant verotoxin subunits hisVT1 B,hisVT2B, pMaIVT1 A and pMa1VT2A(BamHI). Following three or moreimmunizations, IgY was purified from egg yolks by PEG fractionation.Specific antibody response was detected by ELISA, using microtiterplates coated with VT1 or VT2 holotoxin, IgY as primary antibody andalkaline phosphatase: anti-IgY as the secondary antibody. The validityof each ELISA assay was demonstrated with a positive control using rVTIgY and negative controls using Preimmune (PI) IgY.

[0156] These results for this embodiment showed relatively strongbinding of VTIA-G IgY and VT1A-Q IgY to the homologous toxin rVT1 , withtiters of 1:6000 and 1:1200 respectively. However, there was essentiallyno cross reactivity of VT2A-G IgY and VT2A-Q IgY to VT1 holotoxin.VT1A-G IgY and VT1A-Q IgY cross reacted strongly to rVT2; both gave atiter of 1:6000 against rVT2. However, the signal from VT1A-Q IgY wasmuch stronger at the higher concentrations. In contrast, homologousVT2A-Q IgY reactivity to rVT2 gave a much weaker response with a titerof 1:250 and VT2A-G IgY did not react over PI levels.

[0157] For VT1B, significant binding of, VT1B-Q IgY and VT1B-G to VT1holotoxin with titers of 1:2500 and 1:500, respectively was observed.The binding of VT1B-G IgY and VT1B-Q IgY to rVT1 was similar with titersof 1:500 each. Heterologous VT2B-G IgY bound poorly with a titer of1:100 while VT2B-Q IgY had a high titer of 1:1:2500 to rVT1 . There wasmoderate cross-reactivity of VT1B-G IgY and VT1B-Q IgY to VT2, with bothgiving titers of 1:500 and 2500, respectively. In addition, strongreactivity with a titer of 2500 was seen using homologous VT2B-Q IgY toVT2, while VT2B-G IgY showed no significant binding at 1:100.

[0158] Overall, these results indicated that the anti-VT1 A, anti-VT1 Band anti-VT2B of the present invention react with both VT1 and VT2(i.e., they cross react). However, anti-VT2A reacts only with VT2holotoxin.

[0159] B. Use of Antibodies

[0160] It is not intended that antibodies produced against one toxinwill only be used against that toxin. It is contemplated that antibodiesdirected against one toxin may be used as an effective therapeuticagainst one or more toxin(s) produced by other E. coli serotypes, orother toxin producing organisms (e.g., Shigella, Bacillus cereus,Staphylococcus aureus, Streptococcus mutans, Acinetobactercalcoaceticus, Pseudomonas aeruginosa, other Pseudomonas species, Vibriospecies, Clostridium species, etc.). It is further contemplated thatantibodies directed against the portion of the toxin which binds tomammalian membranes can also be used against other organisms. It iscontemplated that these membrane binding domains are producedsynthetically and used as immunogens.

[0161] III. Obtaining Antibodies From Non-Mammals

[0162] A preferred embodiment of the method of the present invention forobtaining antibodies involves immunization. However, it is alsocontemplated that antibodies may be obtained from non-mammals withoutimmunization. In the case where no immunization is contemplated, thepresent invention may use non-mammals with preexisting antibodies totoxins as well as non-mammals that have antibodies to whole organisms byvirtue of reactions with the administered antigen. An example of thelatter involves immunization with synthetic peptides or recombinantproteins sharing epitopes with whole organism components.

[0163] In a preferred embodiment, the method of the present inventioncontemplates immunizing non-mammals with bacterial toxin(s). It is notintended that the present invention be limited to any particular toxin.In one embodiment, toxins from all E. coli serotypes are contemplated asimmunogens.

[0164] A particularly preferred embodiment involves the use of bacterialtoxin protein or fragments of toxin proteins produced by molecularbiological means (i.e., recombinant toxin proteins). In a preferredembodiment, the immunogen comprises recombinant VT1 and/or VT2.

[0165] When immunization is used, the preferred non-mammal is from theclass Aves. All birds are contemplated (e.g., duck, ostrich, emu,turkey, etc.). A preferred bird is a chicken. Importantly, chickenantibody does not fix mammalian complement (See H. N. Benson et al., J.Immunol. 87:616 [1961]). Thus, chicken antibody will normally not causea complement-dependent reaction (A.A. Benedict and K. Yamaga,“Immunoglobulins and Antibody Production in Avian Species,” inComparative Immunology (J. J. Marchaloni, ed.), pp. 335-375, Blackwell,Oxford [1966]). Thus, the preferred antitoxins of the present inventionwill not exhibit complement-related side effects observed withantitoxins presently known.

[0166] When birds are used, it is contemplated that the antibody will beobtained from either the bird serum or the egg. A preferred embodimentinvolves collection of the antibody from the egg. Laying hens transportimmunoglobulin to the egg yolk (“IgY”) in concentrations equal to orexceeding that found in serum (See R. Patterson et al., J. Immunol.89:272 (1962); and S.B. Carroll and B.D. Stollar, J. Biol. Chem. 258:24[1983]). In addition, the large volume of egg yolk produced vastlyexceeds the volume of serum that can be safely obtained from the birdover any given time period. Finally, the antibody from eggs is more pureand more homogeneous; there is far less non-immunoglobulin protein (ascompared to serum) and only one class of immunoglobulin is transportedto the yolk.

[0167] When considering immunization with toxins, one may considermodification of the toxins to reduce the toxicity. In this regard, it isnot intended that the present invention be limited by immunization withmodified toxin. Unmodified (“native”) toxin is also contemplated as animmunogen.

[0168] It is also not intended that the present invention be limited bythe type of modification—if modification is used. The present inventioncontemplates all types of toxin modification, including chemical andheat treatment of the toxin. In one embodiment, glutaraldehyde treatmentof the toxin is contemplated. In an alternative embodiment, formaldehydetreatment of the toxin is contemplated.

[0169] It is not intended that the present invention be limited to aparticular mode of immunization; the present invention contemplates allmodes of immunization, including subcutaneous, intramuscular,intraperitoneal, and intravenous or intravascular injection, as well asper os administration of immunogen.

[0170] The present invention further contemplates immunization with orwithout adjuvant. As used herein, the term “adjuvant” is defined as asubstance known to increase the immune response to other antigens whenadministered with other antigens. If adjuvant is used, it is notintended that the present invention be limited to any particular type ofadjuvant—or that the same adjuvant, once used, be used all the time.While the present invention contemplates all types of adjuvant, whetherused separately or in combinations, the preferred use of adjuvant is theuse of Complete Freund's Adjuvant followed sometime later withIncomplete Freund's Adjuvant. The invention also contemplates the use offowl adjuvant commercially available from RIBI, as well as Quil Aadjuvant commercially available from Accurate Chemical and ScientificCorporation, and Gerbu adjuvant also commercially available (GmDP; C.C.Biotech Corp.).

[0171] When immunization is used, the present invention contemplates awide variety of immunization schedules. In one embodiment, a chicken isadministered toxin(s) on day zero and subsequently receives toxin(s) inintervals thereafter. It is not intended that the present invention belimited by the particular intervals or doses. Similarly, it is notintended that the present invention be limited to any particularschedule for collecting antibody. The preferred collection time issometime after day 35.

[0172] Where birds are used and collection of antibody is performed bycollecting eggs, the eggs may be stored prior to processing forantibody. It is preferred that eggs be stored at 4° C. for less than oneyear.

[0173] It is contemplated that chicken antibody produced in this mannercan be buffer-extracted and used analytically. While unpurified, thispreparation can serve as a reference for activity of the antibody priorto further manipulations (e.g., immunoaffinity purification).

[0174] IV. Increasing The Effectiveness Of Antibodies

[0175] When purification is used, the present invention contemplatespurifying to increase the effectiveness of both non-mammalian antitoxinsand mammalian antitoxins. Specifically, the present inventioncontemplates increasing the percent of toxin-reactive immunoglobulin.The preferred purification approach for avian antibody is polyethyleneglycol (PEG) separation.

[0176] The present invention contemplates that avian antibody beinitially purified using simple, inexpensive procedures. In oneembodiment, chicken antibody from eggs is purified by extraction andprecipitation with PEG. PEG purification exploits the differentialsolubility of lipids (which are abundant in egg yolks) and yolk proteinsin high concentrations of PEG 8000 (Polson et al., Immunol. Comm. 9:495[1980]). The technique is rapid, simple, and relatively inexpensive andyields an immunoglobulin fraction that is significantly more pure, interms of contaminating non-immunoglobulin proteins than the comparableammonium sulfate fractions of mammalian sera and horse antibodies. Themajority of the PEG is removed from the precipitated chickenimmunoglobulin by treatment with ethanol. Indeed, PEG-purified antibodyis sufficiently pure that the present invention contemplates the use ofPEG-purified antitoxins in the passive immunization of intoxicatedhumans and animals.

[0177] V. Treatment

[0178] The present invention contemplates antitoxin therapy for humansand other animals intoxicated by bacterial toxins. A preferred method oftreatment is by parenteral administration of antitoxin. In particularlypreferred embodiments, IgY of the present invention, capable ofneutralizing both VT1 and VT2 is used.

[0179] A. Dosage Of Antitoxin

[0180] It was noted by way of background that a balance must be struckwhen administering currently available antitoxin which is usuallyproduced in large animals such as horses; sufficient antitoxin must beadministered to neutralize the toxin, but not so much antitoxin as toincrease the risk of untoward side effects. These side effects arecaused by: i) patient sensitivity to foreign (e.g, horse) proteins; ii)anaphylactic or immunogenic properties of non-immunoglobulin proteins;iii) the complement fixing properties of mammalian antibodies; and/oriv) the overall burden of foreign protein administered. It is extremelydifficult to strike this balance when, as noted above, the degree ofintoxication (and hence the level of antitoxin therapy needed) can onlybe approximated.

[0181] The present invention contemplates significantly reducing sideeffects so that this balance is more easily achieved. Treatmentaccording to the present invention contemplates reducing side effects byusing PEG-purified antitoxin from birds.

[0182] In one embodiment, the treatment of the present inventioncontemplates the use of PEG-purified antitoxin from birds. The use ofyolk-derived, PEG-purified antibody as antitoxin allows for theadministration of: 1) non (mammalian)-complement-fixing, avian antibody;2) a less heterogeneous mixture of non-immunoglobulin proteins; and 3)less total protein to deliver the equivalent weight of active antibodypresent in currently available antitoxins. The non-mammalian source ofthe antitoxin makes it useful for treating patients who are sensitive tohorse or other mammalian sera.

[0183] As is true in cases of botulism, the degree of an individual'sexposure to E. coli toxin and the prognosis are often difficult toassess, and depend upon a number of factors (e.g., the quantity ofcontaminated food ingested, the toxigenicity and serotype of E. colistrain ingested, etc.). Thus, the clinical presentation of a patient isusually a more important consideration than a quantitative diagnostictest, for determination of dosage in antitoxin administration. Indeed,for many toxin-associated diseases (e.g., botulism, tetanus, diphtheria,etc.), there is no rapid, quantitative test to detect the presence ofthe toxin or organism. Rather, these toxin-associated diseases aremedical emergencies which mandate immediate treatment. Confirmation ofthe etiologic agent must not delay the institution of therapy, as thecondition of an affected patient may rapidly deteriorate. In addition tothe initial treatment with antitoxin, subsequent doses may be indicated,as the patient's disease progresses. The dosage and timing of thesesubsequent doses is dependent upon the signs and symptoms of disease ineach individual patient.

[0184] It is contemplated that the administration of antitoxin to anaffected individual would involve an initial injection of anapproximately 10 ml dose of immune globulin (with less thanapproximately 1 gram of total protein). In one preferred embodiment, itis contemplated that at least 50% of the initial injection comprisesimmune globulin. It is also contemplated that more purified immuneglobulin be used for treatment, wherein approximately 90% of the initialinjection comprises immune globulin. When more purified immune globulinis used, it is contemplated that the total protein will be less thanapproximately 100 milligrams. It is also contemplated that additionaldoses be given, depending upon the signs and symptoms associated with E.coli verotoxin disease progression.

[0185] B. Delivery Of Antitoxin

[0186] Although it is not intended to limit the route of delivery, thepresent invention contemplates a method for antitoxin treatment ofbacterial intoxication in which delivery of antitoxin is parenteral ororal.

[0187] In one embodiment, antitoxin is parenterally administered to asubject in an aqueous solution. It is not intended that the parenteraladministration be limited to a particular route. Indeed, it iscontemplated that all routes of parenteral administration will be used.In one embodiment, parenteral administration is accomplished viaintramuscular injection. In an alternative embodiment, parenteraladministration is accomplished via intravenous injection.

[0188] In another embodiment, antitoxin is delivered in a solid form(e.g., tablets). In an alternative embodiment antitoxin is delivered inan aqueous solution. When an aqueous solution is used, the solution hassufficient ionic strength to solubilize antibody protein, yet is madepalatable for oral administration. The delivery solution may also bebuffered (e.g., carbonate buffer, pH 9.5) which can neutralize stomachacids and stabilize the antibodies when the antibodies are administeredorally. In one embodiment the delivery solution is an aqueous solution.In another embodiment the delivery solution is a nutritional formula.Preferably, the delivery solution is infant or a dietary supplementformula (eg., Similac®, Ensured), and Enfamil®V). Yet another embodimentcontemplates the delivery of lyophilized antibody encapsulated ormicroencapsulated inside acid-resistant compounds.

[0189] Methods of applying enteric coatings to pharmaceutical compoundsare well known to the art (companies specializing in the coating ofpharmaceutical compounds are available; for example, The Coating Place[Verona, Wisc.] and AAI [Wilmington, N.C.]). Enteric coatings which areresistant to gastric fluid and whose release (i.e., dissolution of thecoating to release the pharmaceutical compound) is pH dependent arecommercially available (for example, the polymethacrylates Eudragit® Land Eudragit® S [Röhm Tech Inc., Malden, Mass.]). Eudragit® S is solublein intestinal fluid from pH 7.0; this coating can be used tomicroencapsulate lyophilized antitoxin antibodies and the particles aresuspended in a solution having a pH above or below pH 7.0 for oraladministration. The microparticles will remain intact and undissolveduntil they reached the intestines where the intestinal pH would causethem to dissolve thereby releasing the antitoxin.

[0190] The invention contemplates a method of treatment which can beadministered for treatment of acute intoxication. In one embodiment,antitoxin is administered orally in either a delivery solution or intablet form, in therapeutic dosage, to a subject intoxicated by thebacterial toxin which served as immunogen for the antitoxin. In anotherembodiment of treatment of acute intoxication, a therapeutic dosage ofthe antitoxin in a delivery solution, is parenterally administered.

[0191] The invention also contemplates a method of treatment which canbe administered prophylactically. In one embodiment, antitoxin isadministered orally, in a delivery solution, in therapeutic dosage, to asubject, to prevent intoxication of the subject by the bacterial toxinwhich served as immunogen for the production of antitoxin. In anotherembodiment, antitoxin is administered orally in solid form such astablets or as microencapsulated particles. Microencapsulation oflyophilized antibody using compounds such as EudragitqD (Rohm GmbH) orpolyethylene glycol, which dissolve at a wide range of pH units, allowsthe oral administration of solid antitoxin in a liquid form (i.e., asuspension) to recipients unable to tolerate administration of tablets(e.g., children or patients on feeding tubes). In one preferredembodiment the subject is a child. In another embodiment, antibodyraised against whole bacterial organism is administered orally to asubject, in a delivery solution, in therapeutic dosage. In yet anotherpreferred embodiment of prophylactic treatment, a therapeutic dosage ofthe antitoxin in a delivery solution, is parenterally administered.

[0192] VI. Multivalent Vaccines Against E. coli Strains

[0193] The invention contemplates the generation of multivalent vaccinesfor the protection of an organism (particularly humans) against severalE. coli strains. Of particular interest is a vaccine which stimulatesthe production of a humoral immune response to E. coli O157:H7, 026:H11, O113:H21, O91:H21, and O1 l:NM, in humans. The antigens comprising thevaccine preparation may be native or recombinantly produced toxinproteins from the E. coli serotypes listed above. When native toxinproteins are used as immunogens they are generally modified to reducethe toxicity. It is contemplated that glutaraldehyde-modified toxinproteins will be used. In an alternative embodiment, isformaldehyde-modified toxin proteins will be used.

[0194] The invention contemplates that recombinant E. coli verotoxinproteins be used in conjunction with either native toxins or toxoidsfrom other organisms as antigens in a multivalent vaccine preparation Itis also contemplated that recombinant E. coli toxin proteins be used inthe multivalent vaccine preparation.

[0195] VII. Detection Of Toxin

[0196] The invention contemplates detecting bacterial toxin in a sample.The term “isample” in the present specification and claims is used inits broadest sense. On the one hand it is meant to include a specimen orculture (e.g., microbiological cultures). On the other hand, it is meantto include both biological and environmental samples.

[0197] Biological samples may be animal, including human, fluid, solid(e.g., stool) or tissue, as well as liquid and solid food and feedproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Biological samples may be obtained from all ofthe various families of common domestic animals, including but notlimited, to bovines (e.g, cattle), ovines (e.g., sheep), caprines (e.g.,goats), porcines (e.g., swine), equines (e.g., horses), canines (e.g.,dogs), felines (e.g., cats), lagamorphs (e.g., rabbits), aves (e.g.,chickens, ducks, geese, etc.), and rodents (e.g., mice), etc. It is alsointended that samples may be obtained from feral or wild animals,including, but not limited to, such animals as ungulates (e.g., deer),bear, fish, lagamorphs, rodents, etc.

[0198] Environmental samples include environmental material such assurface matter, soil, water and industrial samples, as well as samplesobtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention.

[0199] The invention contemplates detecting bacterial toxin by acompetitive immunoassay method that utilizes recombinant toxin VT1 andtoxin VT2 proteins, antibodies raised against recombinant bacterialtoxin proteins. A fixed amount of the recombinant toxin proteins areimmobilized to a solid support (e.g., a microtiter plate) followed bythe addition of a biological sample suspected of containing a bacterialtoxin. The biological sample is first mixed with affinity-purified orPEG fractionated antibodies directed against the recombinant toxinprotein. A reporter reagent is then added which is capable of detectingthe presence of antibody bound to the immobilized toxin protein. Thereporter substance may comprise an antibody with binding specificity forthe antitoxin attached to a molecule which is used to identify thepresence of the reporter substance. If toxin is present in the sample,this toxin will compete with the immobilized recombinant toxin proteinfor binding to the anti-recombinant antibody thereby reducing the signalobtained following the addition of the reporter reagent. A control isemployed where the antibody is not mixed with the sample. This gives thehighest (or reference) signal.

[0200] The invention also contemplates detecting bacterial toxin by a“sandwich” immunoassay method that utilizes antibodies directed againstrecombinant bacterial toxin proteins. Affinity-purified antibodiesdirected against recombinant bacterial toxin proteins are immobilized toa solid support (e.g., microtiter plates). Biological samples suspectedof containing bacterial toxins are then added followed by a washing stepto remove substantially all unbound antitoxin. The biological sample isnext exposed to the reporter substance, which binds to antitoxin and isthen washed free of substantially all unbound reporter substance. Thereporter substance may comprise an antibody with binding specificity forthe antitoxin attached to a molecule which is used to identify thepresence of the reporter substance. Identification of the reportersubstance in the biological tissue indicates the presence of thebacterial toxin.

[0201] It is also contemplated that bacterial toxin be detected bypouring liquids (e.g., soups and other fluid foods and feeds includingnutritional supplements for humans and other animals) over immobilizedantibody which is directed against the bacterial toxin. It iscontemplated that the immobilized antibody will be present in or on suchsupports as cartridges, columns, beads, or any other solid supportmedium. In one embodiment, following the exposure of the liquid to theimmobilized antibody, unbound toxin is substantially removed by washing.The liquid is then exposed to a reporter substance which detects thepresence of bound toxin. In a preferred embodiment the reportersubstance is an enzyme, fluorescent dye, or radioactive compoundattached to an antibody which is directed against the toxin (i.e., in a“sandwich” immunoassay). It is also contemplated that the detectionsystem will be developed as necessary (e.g., the addition of enzymesubstrate in enzyme systems; observation using fluorescent light forfluorescent dye systems; and quantitation of radioactivity forradioactive systems).

EXPERIMENTAL

[0202] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0203] In the disclosure which follows, the following abbreviationsapply: rVT (recombinant verotoxin); ° C. (degrees Centigrade); rpm(revolutions per minute); BSA (bovine serum albumin); ELISA(enzyme-linked immunosorbent assay); Ig (immunoglobulin); IgG(immunoglobulin G); IgY (immunoglobulin Y); IP (intraperitoneal); SC(subcutaneous); H_(2O)(water); HCl (hydrochloric acid); LD₁₀₀ (lethaldose for 100% of experimental animals); LD₅₀ (lethal dose for 50% ofexperimental animals); EU (endotoxin unit); aa (amino acid); HPLC (highperformance liquid chromatography); Kda and kd (kilodaltons); gm and g(grams); μg (micrograms); mg (milligrams); ng (nanograms); μl(microliters); ml (milliliters); 1 (liter); mm (millimeters); nm(nanometers); μm (micrometer); ×g and ×g (times gravity); M (molar); mM(millimolar); MW (molecular weight); sec (seconds); min(s)(minute/minutes); hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl(sodium chloride); NACO₃ (sodium carbonate); OD₂₈₀ (optical density at280 nOm); OD₆₀₀ (optical density at 600 um); PAGE (polyacrylamide gelelectrophoresis); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gelelectrophoresis); plysS and plysE (genes encoding T7 lysozyme); IDA(iminodiacetic acid) resin; PBS (phosphate buffered saline [150 mM NaCl,10 mM sodium phosphate buffer, pH 7.2]); PEG (polyethylene glycol); SDS(sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); w/v(weight to volume); v/v (volume to volume); Amicon (Amicon, Inc.,Beverly, Mass.); Amresco (Amresco, Inc., Solon, OH); ATCC (American TypeCulture Collection, Rockville, Md.); BBL (Baltimore BiologicsLaboratory, (a division of Becton Dickinson), Cockeysville, Md.); BectonDickinson (Becton Dickinson Labware, Lincoln Park, N.J.); BioRad(BioRad, Richmond, Calif.); Biotech (C-C Biotech Corp., Poway, Calif.);Charles River (Charles River Laboratories, Wilmington, Mass.); Falcon(e.g., Baxter Healthcare Corp., McGaw Park, Ill. and Becton Dickinson);Fisher Biotech (Fisher Biotech, Springfield, N.J.); GIBCO (Grand IslandBiologic Company/BRL, Grand Island, N.Y.); Mallinckrodt (a division ofBaxter Healthcare Corp., McGaw Park, Ill.); Millipore (Millipore Corp.,Marlborough, Mass.); New England Biolabs (New England Biolabs, Inc.,Beverly, Mass.); Novagen (Novagen, Inc., Madison, Wisc.); Pharmacia(Pharmacia, Inc., Piscataway, N.J.); Qiagen (Qiagen, Chatsworth,Calif.); Showdex (Showa Denko America, Inc., New York, N.Y.); Sigma(Sigma Chemical Co., St. Louis, Mo.); RIBI (RIBI Immunochemical ResearchInc., Hamilton, Mont.); Accurate Chemical and Scientific Corp. (AccurateChemical and Scientific Corp., Hicksville, N.Y.); Kodak (Eastman-Kodak,Rochester, N.Y.); and Sterogene (Sterogene Bioseparations, Inc.,Carlsbad, Calif.); and Stratagene (Stratagene, La Jolla, Calif.).

[0204] When a recombinant protein is described in the specification itis referred to in a short-hand manner by the amino acids in the toxinsequence present in the recombinant protein rounded to the nearest 10.The specification gives detailed construction details for allrecombinant proteins such that one skilled in the art will knowprecisely which amino acids are present in a given recombinant protein.

[0205] The first set of Examples (Examples 1-5) was designed to developan antidote to E. coli O157:H7 verotoxins and evaluate its effectivenessin vitro and in vivo. In the first experiments, high titer verotoxinantibodies were generated in laying hens hyperimmunized with chemicallydetoxified and/or native verotoxins. These Laying hens were immunizedwith either recombinant E. coli O157:H7 VT1 or VT2 (rVTI and rVT2)treated with glutaraldehyde and mixed with adjuvant.

[0206] Next, toxin-reactive polyclonal antibodies were isolated by bulkfractionation from egg yolks pooled from hyperimmunized hens. Largequantities of polyclonal antibodies (IgY) were harvested from resultingeggs using a two-step polyethylene glycol fractionation procedure.

[0207] Third, the immunoreactivity and yields of VT IgY were analyzed byanalytical immunochemical methods (e.g., enzyme immunoassay (EIA) andWestern blotting). EIA and Western blot analysis showed that theresulting egg preparations contained high titer IgY that reacted withboth the immunizing and the heterologous toxins (i.e., rVT1 IgY reactedagainst both rVT1 and rVT2, and vice versa).

[0208] Fourth, VT neutralization potency was analyzed in vitro using aVero cytotoxicity assay. Vero cytotoxicity of rVTI and rVT2 could becompletely inhibited by VT IgY. These antibodies also demonstratedsubstantial verotoxin cross-neutralization.

[0209] Fifth, the efficacy of passively administered avian verotoxinantibodies in preventing the lethal effects of verotoxin poisoning wasassessed in a mouse disease model. Toxin neutralizing antibodies wereadministered by parenteral dosing regimens to assess the most effectivestrategy for therapeutic intervention. Efficacy of verotoxin antibodieswas demonstrated using multiple murine disease models. In theseexperiments, antibodies prevented both the morbidity and lethality ofhomologous and heterologous toxins using a toxin/antitoxin premixformat; mice infected orally with a lethal dose of viable E. coliO157:H7 were protected from both morbidity and lethality when treatedparenterally four hours post-infection with either rVT1 or rVT2antibodies; and mice given a lethal dose of E. coli O91:H21 (aparticularly virulent strain which only produces VT2c, a VT2 structuralvariant) and treated parenterally up to 10 hours later with rVT1 IgYadministered parenterally were protected from both morbidity andlethality.

[0210] Sixth, verotoxin clones were constructed and expressed, includingHis-tagged and MBP fusions, as well as vectors for expression of VT1Band VT2B subunits without His-tags. Next, fermentation cultures (i. e.,large scale preparations) of the expressed VT2B protein were produced.The expressed verotoxin subunits were used as immunogens in hens andrabbits, the titers determined by quantitative ELISA methods developedas described in the following examples. Finally, the protective abilityof the rabbit and chicken IgY antisera were tested in in vivo challengesin mice.

EXAMPLE 1 TOXIN ANALYSIS AND IMMUNIZATION

[0211] Purified recombinant E. coli O157:H7 verotoxins, rVT1 and rVT2,were obtained from Denka Sieken Co., Ltd. (Tokyo, Japan). Recombinantproteins were then purified by ammonium sulfate precipitation, ionexchange chromatography on DEAE Sephacryl and hydroxyapatite, and gelfiltration chromatography by the supplier. Toxin genes were isolated,inserted into expression plasmids, and expressed in E. coli . Uponreceipt, toxins were analyzed to verify identity, purity and toxicity,as described below.

[0212] A. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE).

[0213] Samples of each toxin (2 jig) were heat-denatured in a buffercontaining SDS and β-mercaptoethanol followed by electrophoresis on 420%gradient gels (Bio-Rad, Richmond, Calif.). Resolved polypeptide bandswere visualized using the silver stain procedure of C.R. Merril, et al.,“Ultrasensitive stain for proteins in polyacrylamide gels shows regionalvariation in cerebrospinal fluid proteins,” Science 211: 1437-1438(1981).

[0214] VT1 and VT2 are each composed of subunit A and multiple copies ofsubunit B. Subunit A is often nicked into fragments A1 and A2 which arelinked by a disulfide bridge. As shown in FIG. 1, when separated bySDS-PAGE in the presence of β-mercaptoethanol, rVT1 resolved into 3bands that corresponded to subunit A (˜31 Kda), fragment A1 (˜27 Kda)and a mixture of subunit B and fragment A2 (˜4 Kda). Similarly, rVT2resolved into subunit A (˜33 Kda), fragment Al (˜27 Kda) and a mixtureof subunit B and fragment A2 (˜8 Kda) (FIG. 1). In this Figure, rVT1 isin lane 1, and rVT2 is in lane 2; the positions of molecular weightmarkers (Kda) are shown at the left. rVT component polypeptides areidentified at the right.

[0215] These results are consistent with previous reports of VT1 and VT2purified from naturally occurring toxigenic strains (V. V. Padhye etal., “Purification and Physicochemical Properties of a Unique Vero CellCytotoxin From Escherichia coli O157:H7,” Biochem. Biophys. Res.Commun., 139: 424-430 [1986]; and F. B. Kittel et al., “Characterizationand inactivation of verotoxin 1 produced by Escherichia coli O157:H7,”J. Agr. Food Chem., 39: 141-145 [1991]).

[0216] B. High Performance Liquid Chromatography (HPLC).

[0217] Chromatography was performed at room temperature (RT) underisocratic conditions using a Waters 510 HPLC pump. Eluted protein wasmeasured using a Waters 490E programmable multi-wavelength detector(Millipore Corp., Milford, Mass.). The VT's were separated on an 8×300mm (ID) Shodex KW803 column, using 10 mM sodium phosphate, 0.15 M NaCl,pH 7.4 (PBS) as the mobile phase at a flow rate of 1 ml/min.

[0218] The purity of non-denatured rVT's was assessed by HPLC. As shownin the chromatographs in FIG. 2, each toxin eluted at approximately 10min. as a single absorbance peak at 280 nm. In this Figure, Panel Ashows the HPLC results for rVTI, and Panel B shows the HPLC results forrVT2. By integration of the area under each peak, the rVT's were shownto be >99% pure.

[0219] C. Vero Cell Cytotoxicity Assay.

[0220] Cytotoxic activity of rVT1 and rVT2 was assessed using modifiedprocedures of Padhye, et al. (V. V. Padhye et al, “Purification andPhysicochemical Properties of a Unique Vero Cell Cytotoxin FromEscherichia coli O157:H7,” Biochem. Biophys. Res. Commun., 139: 424-430[1986]), and McGee, et al., (Z. A. McGee, et al., “Local induction oftumor necrosis factor as molecular mechanism of mucosal damage bygonococci,” Microbial Pathogenesis 12: 333-341 [1992]). Microtiterplates (96 well, Falcon, Microtest III) were inoculated withapproximately 1×104 Vero cells (ATCC, CCL81) per well (100 ptl) andincubated overnight at 37° C. in the presence of 5% CO₂ to form Verocell monolayers. rVTI and rVT2 solutions were serially diluted in Medium199 supplemented with 5% fetal bovine serum (Life Technologies, GrandIsland, N.Y.), added to each well of the microtiter plates and incubatedat 37° C. for 18-24 hrs. Adherent (viable) cells were stained with 0.2%crystal violet (Mallinckrodt) in 2% ethanol. Excess stain was rinsedaway and the stained cells were solubilized by adding 100 tl of 1% SDSto each well. Absorbance of each well was measured at 570 nm, and thepercent cytotoxicity of each test sample was calculated using thefollowing formula:

% Vero Cytotoxicity=[1−(Absorbance Sample/Absorbance Control)]×100

[0221] To determine whether the rVT's possessed potency equivalent topublished cytotoxicity values, a Vero cell cytotoxicity assay wasperformed (FIG. 3). Between 0.01-10,000 pg of either rVT1 or rVT2 wasadded to Vero cells. The amounts of rVT causing 50% cell death (CD₅₀),as calculated by second degree polynomial curve fitting were 0.97 pg and1.5 pg, for rVT1 and rVT2, respectively. These results are consistentwith CD₅₀ values reported previously for naturally occurring VT1 and VT2in the range 1-35 pg and 1-25 pg, respectively (M. Petric et al.,Purification and biological properties of Escherichia coliverocytotoxin,” FEMS Microbiol. Lett., 41: 63-68 [1987]; V. L. Tesh, etal., “Comparison of relative toxicities of Shiga-Like toxins Type I andType II for mice,” Infect. Immun., 61: 3392-3402 [1993]; N. Dickie etal., “Purification of an Escherichia coli Serogroup O157:H7 verotoxinand its detection in North American hemorrhagic colitis isolates,” J.Clin. Microbiol., 27: 1973-1978 [1989]; and U. Kongmuang, et al., “Asimple method for purification of Shiga or Shiga-Like toxin fromShigella dysenteriae and Escherichia coli O157:H7 by immunoaffinitychromatography,” FEMS Microbiol. Lett., 48: 379-383 [1987]). It has beenobserved that toxicity is lost with storage, explaining why higheramounts of toxin were used in the neutralization assays described below.

[0222] D. Mouse Lethal Dose Determination.

[0223] To verify rVT1 and rVT2 toxicity, male (20-22 g) CD-1 mice wereinjected intraperitoneally with varying amounts of rVT1 or rVT2 in 200JL phosphate buffer. Doses were selected based on published LD₅₀ valuesfor VT1 and VT2 in CD-1 mice. To minimize the sacrifice of live animals,a full statistical toxin LD₅₀ was not determined. Mice were observed formorbidity and mortality over 7-day period.

[0224] Further confirmation of rVT toxicity was obtained from mouselethality experiments (Tables 2 and 3). Mice were injectedintraperitoneally with varying amounts of either rVTI or rVT2 andobserved 7 days for mortality. Within 72-120 hrs. post-injection, all ofthe mice died from 100 ng of rVT1 or 10 ng of rVT2, respectively. Thislethality study served as a verification of expected toxicity but not asa statistical determination of LD50. Nonetheless, these results wereconsistent with toxicity studies which reported LD,, values in CD-1 miceof 0.4-2.0 μg for purified VT1 and 0.001-1.0 μg for purified VT2 (V. L.Tesh, et al, “Comparison of relative toxicities of Shiga-Like toxinsType I and Type II for mice,” Infect. Immun., 61: 3392-3402 [1993]; andA. D. O'Brien, and G. D. LaVeck, “Purification and characterization ofShigella dysenteriae 1-like toxin produced by Escherichia coli,” Infect.Immun. 40: 675-683 [1983]). TABLE 2 Lethality of rVT1 in CD-1 Mice ngVT1 Injected Survivors/Total Hours Post-Injection 100 7/7 24 ± 2 5/7 48± 2 0/7 72 ± 2 10 7/7 24 ± 2 7/7 48 ± 2 7/7 72 ± 2 1.0 6/6 24 ± 2 6/6 48± 2 6/6 72 ± 2

[0225] TABLE 3 Lethality of rVT2 in CD-1 Mice ng VT2 InjectedSurvivors/Total Hours Post-Injection 10 3/6 48 ± 2 2/6 72 ± 2 0/6 120 ±2  1.0 5/6 48 ± 2 4/6 72 ± 2 0/6 120 ± 2  0.1 6/6 48 ± 2 6/6 72 ± 2 6/6120 ± 2 

[0226] The recombinant toxins used in these studies thus appeared tocontain protein components and toxicities consistent with literaturereports for native toxins. Based on these structural and functionalanalyses, the rVT's were considered suitable as antigens to generatespecific avian antibodies.

[0227] E. Antigen Preparation.

[0228] Lyophilized samples, rVTI and rVT2 were received and each wasreconstituted with 2.5 mL of deionized water to a final concentration of100 μg/ml in phosphate buffer. To form a toxoid, the solutions were thentreated with 0.4% glutaraldehyde (Mallinckrodt) at 4° C. overnight andstored at −20° C. thereafter. When needed, toxoid was thawed and mixed5:1 (volume:volume) with 5 μg GERBU adjuvant (C. C. Biotech Corporation,Poway, CA). White Leghorn laying hens were injected subcutaneously with25 jig of either rVT1 or rVT2 toxoid in adjuvant at 2-3 week intervals.

EXAMPLE 2 PEG EXTRACTION OF EGG YOLK ANTIBODY

[0229] Hyperimmune eggs were collected after 3 immunizations withtoxoid. Egg yolks were separated from whites, pooled according to theirimmunogen group and blended with 4 volumes of 10 mM sodium phosphate,150 mM NaCl, pH 7.4 (PBS). Polyethylene glycol 8000 (PEG) (Amresco,Solon, OH) was then added to a final concentration of 3.5% and themixture centrifuged at 10,000×g for 10 min. to remove the precipitatedlipid fraction. IgY-rich supernatant was filtered through cheeseclothand PEG was again added to a final concentration of 12%. The solutionwas centrifuged as above and the resulting supernatant discarded. TheIgY pellet was then dissolved in PBS to either the original (1×PEG IgY)or ¼ of the original (4×PEG IgY) yolk volume, filtered through a 0.45 μmembrane and stored at 4° C.

EXAMPLE 3 ANTITOXIN IMMUNOASSAYS

[0230] A. Enzyme Immunoassay (EIA).

[0231] EIA was used to monitor antibody responses during theimmunization course. Wells of 96-well Pro-Bind microtiter plates(Falcon, through Scientific Products, McGaw Park, Ill.) were each coatedovernight with 100 μl of PBS containing 1 μg/ml rVT's (not toxoid) at2-8° C. Wells were washed 3 times with PBS containing 0.05% Tween-20(PBS-T) to remove unbound antigen, and the remaining protein bindingsites were blocked with PBS containing 5 mg/ml BSA for 60 min. at 37° C.IgY, diluted in PBS containing 1 mg/ml BSA and 0.05% Tween-20 was thenadded to the wells and incubated for 1 hr. at 37° C. Wells were washedas before to remove unbound primary antibody and incubated for 1 hr. at37° C. with alkaline phosphatase-conjugated rabbit-anti-chicken IgG(Sigma Chemical Company, St. Louis, Mo.) diluted 1:1000 in PBS-T. Wellswere again washed and 1 mg/ml p-nitrophenyl phosphate (Sigma ChemicalCompany, St. Louis, Mo.) in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 was addedand allowed to incubate at RT. Phosphatase activity was detected byabsorbance at 410 nm using a Dynatech MR700 microtiter plate reader.

[0232] Laying Leghorn hens were immunized as described above (Example 1,part E), using glutaraldehyde-treated rVT's. Following severalimmunizations, eggs were collected and IgY harvested by PEGfractionation. FIGS. 4 and 5 show rVTI or rVT2 specific antibodyresponses detected using EIA at dilutions of the original yolk IgYconcentration of 1:30,000 and 1:6,000, respectively. IgY fractionatedsimilarly from unimmunized hens (i.e., preimmune antibody) did not reactwith either antigen at test dilutions above 1:50. Although these EIAresults indicate significant antibody responses, prior experience withother toxin antigens has shown that optimization of immunizationregimens, including increasing the amount of antigen, can yield titersin excess of 1:100,000 (B. S. Thalley, et al.,“Development of an AvianAntitoxin to Type A Botulinum Neurotoxin,” in Botulinum and TetanusNeurotoxins: Neurotransmission and Biomedical Aspects, B. R. DasGupta,(ed.) [Plenum Press, New York, 1993] pp. 467-472). As may be expecteddue to their structural homology and consistent with previous reports(e.g., V. V. Padhye et al., “Production and characterization ofmonoclonal antibodies to verotoxins 1 and 2 from Escherichia coliO157:H7,” J Agr. Food Chem., 39: 141-145 [1989]; S. C. Head et al.,“Purification and characterization of verocytotoxin 2,” FEMS Microbiol.Lett., 51: 211-216 [1988]; and N. C. Strockbine et al.,“Characterization of Monoclonal Antibodies against Shiga-Like Toxin fromEscherichia coli,” Infect. Immun., 50: 695-700 [1985]), FIGS. 4 and 5also demonstrate that antibodies generated against one toxincross-reacted in vitro with the other toxin.

[0233] B. Western Blot Analysis.

[0234]

[0235] Western blots (FIG. 6) performed to determine the reactivity ofrVT antibodies against constituent VT polypeptides showed that rVT1 andrVT2 antibodies reacted with subunit A and fragment Al of either toxin,and with subunit B and fragment A2 of rVT1 only. In this Figure, Panel Acontains preimmune IgY, Panel B contains rVT1 IgY, and Panel C containsrVT2 IgY. Lane 1 in each panel contains rVT1 (2 jig) and lane 2 containsrVT2 (2 μg). Preimmune IgY was largely nonreactive to either rVT. BothrVT IgY preparations, however, failed to react with subunit B andfragment A2 of rVT2. Some explanations for this lack of measurablereactivity might include poor immunogenicity, denaturation of theimmunogen during glutaraldehyde treatment, loss of conformationalepitopes due to detergent or reducing agent, or poor transfer tonitrocellulose.

[0236] To resolve the high and low molecular weight components, 2 μgeach of rVT1 and rVT2 were separated by SDS-PAGE (described above) andthen transferred to nitrocellulose paper using the Milliblot-SDE system(Millipore, Medford, Mass.) according to the manufacturer'sinstructions. Nitrocellulose strips were stained temporarily withPonceau S (Sigma Chemical Company, St. Louis, Mo.) to visualize thepolypeptides and then blocked overnight in PBS containing 5% dry milk.Each strip was agitated gently in IgY diluted in PBS-T for 2 hrs. at RT.Strips were each washed with three changes of PBS-T to remove unboundprimary antibody and incubated for 2 hrs. at RT with goat anti-chickenalkaline phosphatase (Kirkegaard and Perry, Gaithersburg, Md.) diluted1:500 in PBS-T containing 1 mg/ml BSA. The blots were washed as beforeand rinsed in 50 mM Na₂CO₃, pH 9.5. Strips were submerged inalkaline-phosphatase substrate(5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (Kirkegaardand Perry) until sufficient signal was observed. Color development wasstopped by flooding the blots with water.

EXAMPLE 4 IN VITRO TOXIN NEUTRALIZATION VERO CELL ASSAY

[0237] IgY neutralization of rVT1 and rVT2 was assessed using themodified Vero cytotoxicity assay described above (Example 1, part C).Various concentrations of IgY, diluted in Medium 199 supplemented with5% fetal bovine serum (GIBCO), were mixed with sufficient toxin to cause50% cell death and allowed to incubate at 37° C for 60 minutes. Thesetoxin/antibody mixtures were then added to Vero cell-coated microtiterplate wells according to the procedure described above (Example 1, partC).

[0238] The toxin neutralization capacity of the rVT antibodies wasanalyzed first using a Vero cell toxicity assay. The results in FIG. 7show that rVT1 IgY neutralized completely the cytotoxic activity of rVT1at an endpoint dilution of 1/320. Furthermore, rVT2 IgY neutralized theheterologous rVT1 toxin, but at a higher endpoint concentration.

[0239] In a similar experiment (see FIG. 8), rVT1 and rVT2 antibodieswere each able to neutralize rVT2 at equivalent endpoint dilutions. Thisstrong cross-neutralization correlates with the observed strongcross-reactivity of VT1 IgY with VT2 A seen on Western blots (FIG. 6).These results show that IgY antibodies are able to neutralizeeffectively VT cytotoxicity and that the antibodies can cross-neutralizestructurally-related heterologous toxins.

EXAMPLE 5 TOXIN NEUTRALIZATION: MOUSE ASSAYS

[0240] A. Toxin Challenge Model.

[0241] IgY in PBS was premixed with a lethal dose of toxin (asdetermined above) and injected intraperitoneally into male CD-1 (20-22gm) mice. Mice were observed for a 7-day period for signs ofintoxication such as ruffled fur, huddling and disinclination to move,followed by hind leg paralysis, rapid breathing and death. Untreated,infected mice usually died within 12 hrs. after signs of severe illness(i.e., within 48-72 hrs. post-injection).

[0242] Once it was demonstrated that rVT antibodies were able toneutralize rVT cytotoxicity in vitro, protection experiments were nextperformed in mice. First, animals were challenged with rVT premixed withrVT IgY to determine whether toxin lethality could be neutralized underconditions optimal for antigen/antibody reaction. Tables 4 and 5 showthat antibodies premixed with the homologous toxin (e.g., rVT1 with rVT1IgY) prevented lethality of rVT. Preimmune IgY was unable to neutralizeeither toxin in these studies. TABLE 4 Neutralization of rVT1 Using rVTIgY 100 ng rVT1 Premixed* Survivors/Total p Preimmune Antibody  0/12rVT1 Antibody 12/12 <0.001 rVT2 Antibody 12/12 <0.001

[0243] TABLE 5 Neutralization of rVT2 Using rVT IgY 10 ng rVT2 Premixed*Survivors/Total p Preimmune Antibody  0/12 rVT1 Antibody 12/12 <0.001rVT2 Antibody 12/12 <0.001

[0244] As shown in Tables 4 and 5, antibodies premixed with theheterologous toxin (e.g., rVT2 with rVT1 IgY) also prevented lethalityin vivo. These data are in contrast to previous observations whererabbit polyclonal antibodies generated against either toxin werecross-reactive with the heterologous toxin by EIA and Western blot, butwere unable to neutralize the heterologous toxin in either Vero cellcytotoxicity and mouse lethality assays (S. C. Head, et al.,“Serological differences between verocytotoxin 2 and Shiga-like toxinII,” Lancet ii: 751 [1988]; S. C. Head et al., “Purification andcharacterization of verocytotoxin 2,” FEMS Microbiol. Lett., 51: 211-216[1988]; N. C. Strockbine et al., “Characterization of MonoclonalAntibodies against Shiga-Like Toxin from Escherichia coli,” Infect.Immun., 50: 695-700 [1985]; and V. V. Padhye et al., “Purification andPhysicochemical Properties of a Unique Vero Cell Cytotoxin FromEscherichia coli O157:H7,” Biochem. Biophys. Res. Commun., 139: 424-430[1986]).

[0245] However, Head et al., showed that VT2 B-subunit specificmonoclonal antibodies neutralized VT1 weakly in a Vero cytotoxicityassay (S. C. Head, et al., “Serological differences betweenverocytotoxin 2 and Shiga-like toxin II,” Lancet ii: 751 [1988]). In areport by Donohue-Rolfe, et al., a VT2 B subunit-specific monoclonalantibody neutralized both VT1 and VT2 completely in a Hela cytotoxicityassay (A. Donohue-Rolfe et al., “Purification of Shiga toxin andShiga-like toxins I and II by receptor analog affinity chromatographywith immobilized PI glycoprotein and production of cross reactivemonoclonal antibodies,” Infect. Immun., 57: 3888-3893 [1989]).

[0246] These results showed for the first time completecross-neutralization in Vero cell cytotoxicity and mouse lethalityassays, revealing that VT1 and VT2 do indeed share common neutralizingepitopes. These results may indicate that hens generate differentantibody specificities as compared to mammals, and/or that differencesin immunization methods might have maintained the immunogenicity ofconformational epitopes necessary for cross-neutralization. Nonetheless,this cross-neutralization suggests that IgY antibodies may contain therange of reactivities essential for an effective antitoxin.

[0247] B. Viable organism infection model.

[0248]

[0249] Streptomycin-resistant E. coli O157:H7 (strain 933 cu-rev) or E.coli O91::H21 (strain B2F1) (both kindly provided by Dr. Alison O'Brien,Dept. of Microbiology and Immunology, Uniformed Services University ofthe Health Sciences, Bethesda, Md.) were used in a murine infectionmodel described by Wadolkowski, et al. (E. A. Wadolkowski et al., “Mousemodel for colonization and disease caused by enterohemorrhagicEscherichia coli O157:H7,” Infect. Immun., 58: 2438-2445 [1990]).Organisms were grown in Luria broth and incubated overnight at 37° C. inan Environ Shaker (Lab Line, Melrose Park, Ill.) (T. Maniatis et al.,Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., [1982]). Bacterial suspensions werecentrifuged at 6700 x g for 5 minutes. The resulting pellet was thenwashed twice with sterile PBS and resuspended in sterile 20% (w/v)sucrose. Five to 8 week-old male CD-1 mice were provided drinking watercontaining 5 mg/ml streptomycin sulfate ad libitum for 24 hrs. Food andwater were then withheld for another 16-18 hrs, after which mice werechallenged orally with 10¹⁰ streptomycin-resistant E. coli O157:H7 orO91:H21. Mice were housed individually and permitted food and watercontaining 5 mg/ml streptomycin sulfate. IgY was injectedintraperitoneally at varying times post-infection and animals observedfor both morbidity and mortality for 10 days.

[0250] To monitor bacterial colonization in animals, 1 gram of feces wascollected, homogenized, and plated onto MacConkey agar medium (DifcoLaboratories, Detroit, MI) containing 100 μg/ml streptomycin andincubated at 37° C. as described by Wadolkowski, et al. (E. A.Wadolkowski et al., “Mouse model for colonization and disease caused byenterohemorrhagic Escherichia coli O157:H7,” Infect. Immun., 58:2438-2445 [1990]). The serotype of E. coli O157:H7, 933 cu-rev excretedin feces was confirmed by slide agglutination with O- and H-specificantisera (Difco Laboratories, Detroit, Mich.).

[0251] Kidneys were removed from experimental animals and fixed in 10%buffered neutral formalin. Sections of parafilm-embedded tissue werestained with hematoxylin and eosin (General Medical Laboratories,Madison, Wisc.) and examined by light microscopy. All tissue sectionswere coded to avoid bias before microscopic examination to determinerenal pathology.

[0252] The toxin neutralization ability of rVT IgY was further studiedusing a streptomycin-treated CD-1 mouse infection model. This model waschosen because it produces definitive systemic pathology andreproducible mortality.

[0253] In contrast to previous studies by Wadolkowski, et al. (E. A.Wadolkowski et al., “Acute renal tubular necrosis and death of miceorally infected with Escherichia coli strains that produce Shiga-liketoxin Type II,” Infect. Immun., 58: 3959-3965 [1990]), where mice weregiven subunit-specific monoclonal antibodies prior to infection, themice in this study were inoculated orally with 2×10¹⁰ viable E. coliO157:H7 (strain 933 cu-rev) and treated with rVT IgY 4 hrs. followinginoculation. Fecal cultures showed that 10⁷-10⁸ challenge organisms pergram of feces were shed throughout the course of the experiment, thusconfirming that infection was established. Tables 6 and 7 show thatanimals treated with either rVT1 or rVT2 IgY were protected fromlethality caused by infection (p<0.01 and p<0.001, respectively) andthat preimmune IgY failed to provide protection to the mice. TABLE 6Protection of Mice From E. coli O157:H7 With rVT1 IgY IgY TreatmentSurvivors/Total p Morbidity/Total Preimmune Antibody 0/5  5/5  rVT1Antibody 9/10 <0.01 1/10

[0254] TABLE 7 Protection of Mice From E. coil O157:H7 With rVT2 IgY IgYTreatment Survivors/Total p Morbidity/Total Preimmune 0/6 6/6  AntibodyrVT2 Antibody 10/10 <0.005 0/10

[0255] Renal histopathology (see FIG. 9) of the control (preimmune IgY)animals showed dilation, degeneration and renal tubular necrosis with noglomerular damage. Panel A shows a representative kidney section from amouse treated with preimmune IgY, Panel B shows a representative kidneysection from a mouse treated with rVT1 IgY, and Panel C shows arepresentative kidney section from a mouse treated with rVT2 IgY; all ofthese panels show the results 4 hours after infection. This isconsistent with previous reports showing that renal tubular involvementoccurs predominantly in this streptomycin-treated mouse infection model(E. A. Wadolkowski et al., “Acute renal tubular necrosis and death ofmice orally infected with Escherichia coli strains that produceShiga-like toxin Type II,” Infect. Immun., 58: 3959-3965 [1990]).Importantly, none of the survivors exhibited similar signs of morbiditythough treated with IgY 4 hrs. after infection (see FIG. 9).

[0256] Furthermore, avian antibodies generated against rVT1 were able toprevent both mortality and morbidity in a mouse model where VT2 alone isimplicated in the pathogenesis and lethality of E. coli O157:H7 strain933 cu-rev (E. A. Wadolkowski et al., “Acute renal tubular necrosis anddeath of mice orally infected with Escherichia coli strains that produceShiga-like toxin Type II,1” Infect. Immun., 58: 3959-3965 [1990]).

[0257] To assess the broader utility of the IgY verotoxin antibodies intreating VTEC-associated disease, the mouse infection study wasperformed using a more virulent VTEC serotype known to produce VT2c—astructural variant of VT2—but not VT1 (S. W. Lindgren et al., “Virulenceof enterohemorrhagic Escherichia coli O91:H21 clinical isolates in anorally infected mouse model,” Infect. Immun., 61: 3832-3842 [1993]).

[0258] Mice were inoculated orally with 5×10⁹ E. coli O91:H21 (strainB2F1) and treated subsequently with IgY. Notably, the heterologous rVT1IgY protected strongly against the lethal effects of the VT2c structuralvariant, even when administered as long as 10 hrs. following infection(Table 8). Ten hours was the longest treatment window tested in thisstudy. Only 1 of the 8 animals treated with rVT1 IgY died (p <0.02), andthose that survived showed no signs of renal pathology (i.e., acutebilateral tubular necrosis). It can thus be concluded that rVT1 IgYcompletely neutralized toxicity of VT2c, indicating its potential as atherapeutic for at least one other pathogenic VTEC. TABLE 8 Protectionof Mice From E. coli O91:H21 With rVT1 IgY IgY Treatment Survivors/Totalp Morbidity/Total Preimmune Antibody 0/7 7/7 rVT1 Antibody 7/8 <0.02 1/8

[0259] These Examples highlight several important findings supportingthe feasibility of using verotoxin antitoxin. First, polyclonal IgYgenerated against either VT1 or VT2 from E. coli O157:H7, cross-reactedwith and fully cross-neutralized the toxicity of the non-immunizingtoxin both in vitro and in vivo. Second, recombinant toxins fullyneutralized the toxicity of naturally-occurring toxins produced by E.coli O157:H7 during the course of infection. Third, antibodies generatedagainst rVT1 from E. coli O157:H7 could prevent morbidity and mortalityin mice infected orally with lethal doses of E. coli O91 :H21, aparticularly virulent strain which only produces VT2c, suggesting theirutility in preventing systemic sequelae. Because VT1 is virtuallyidentical to Shiga-toxin (A. D. O'Brien et al., “Shiga and Shiga-liketoxins. Microbial Rev., 51: 206-220 [1987]), VT antibodies may also beuseful in preventing complications stemming from Shigella dysenteriaeinfection. Finally, animals treated with VT IgY were protected againstboth death and kidney damage when treated as long as 10 hrs. afterinfection, supporting the hypothesis that a window for antitoxinintervention exists.

[0260] These studies strongly support the use ofparenterally-administered, toxin-specific IgY as a antitoxin to preventlife-threatening complications associated with E. coli O157:H7 and otherVTEC infections. It is contemplated that this approach would be usefulin preventing HUS and other complications when administered after theonset of bloody diarrhea and before the presentation of systemicdisease.

[0261] The VT IgY developed in these studies were shown to react withand neutralize both recombinant and naturally-occurring VT. The antibodytiters as measured by EIA are indicative of reasonable antibodyproduction in the hen, however much higher production levels can beobtained with larger immunizing doses.

[0262] The results from these Examples clearly demonstrate thefeasibility and provide the experimental basis for development of anavian antidote for E. coli O157:H7 verotoxins suitable for use inhumans. In contrast to previous reports showing that rabbit polyclonalVT1 and VT2 antibodies cross-reacted, but did not cross-neutralize theheterologous toxin in Vero cytotoxicity or in mouse lethality studies(e.g., V. V. Padhye et al., “Production and characterization ofmonoclonal antibodies to verotoxins 1 and 2 from Escherichia coliO157:H7,” J. Agr. Food Chem., 39: 141-145 [1989]; S. C. Head et al.,“Purification and characterization of verocytotoxin 2,” FEMS Microbiol.Lett., 51: 211-216 [1988]; and N. C. Strockbine et al.,“Characterization of monoclonal antibodies against Shiga-like toxin fromEscherichia coli,” Infect. Immun., 50: 695-700 [1985]), these dataprovide the first demonstration of cross-neutralization in vivo.Antibodies against one toxin neutralized completely the heterologoustoxin in both Vero cytotoxicity and mouse lethality assays. Both rVT1and rVT2 antibodies also prevented morbidity (as assessed by renalhistopathology) and mortality in mice infected with lethal doses of E.coli O157:H7—the etiologic agent in 90% of the documented cases ofhemolytic uremic syndrome (HUS) in the U.S. (P. M. Griffin and R. V.Tauxe, “The epidemiology of infections caused by Escherichia coliO157:H7, other enterohemorrhagic E. coli , and the associated hemolyticuremic syndrome,” Epidemiol. Rev., 13: 60 [1990]). With at least twoother VTEC serotypes known to cause HUS, the finding that rVT1antibodies neutralized a VT2 variant produced by E. coli O91:H21suggests that avian polyclonal antibodies may provide an effectiveantidote against other verotoxin-producing E. coli . These data alsoshow for the first time, that antibodies may be administered afterinfection and still protect against morbidity and mortality.

EXAMPLE 6 EXPRESSION OF TOXIN GENES

[0263] The previous Examples clearly showed that avian polyclonalantibodies to recombinant toxins protected animals infected withverotoxigenic E. coli . This Example includes expression of toxin genes(A and B subunits alone and together as whole toxins) in suitableprokaryotic expression systems to achieve high levels of VT antigenproduction.

[0264] The sequence of both toxin genes (VT1 and VT2) has beendetermined (see e.g., M. P. Jackson et al., “Nucleotide sequenceanalysis and comparison of the structural genes for Shiga-like toxin Iand Shiga-like toxin II encoded by bacteriophages from Escherichia coli933,” FEMS Microbiol. Lett., 44:109 [1987]; and Jackson et al., Microb.Pathogen., 2:147-153 [1987]). The genes show similar organization, suchthat the A and B chains of each toxin are juxtaposed in the same 5′ to3′ orientation.

[0265] The coding regions of the A and B subunits of VT-1 are listed inSEQ ID NOS:l and 3, respectively. The corresponding amino acid sequenceof the A and B subunits of the VT-1 toxin are listed in SEQ ID NOS:2 and4, respectively. The coding regions of the A and B subunits of VT-2 arelisted in SEQ ID NOS:5 and 7, respectively. The corresponding amino acidsequence of the A and B subunits of the VT-2 toxin are listed in SEQ IDNOS:6 and 8, respectively. In addition, SEQ ID NOS:9 and 10 list thesequences which direct the expression of a poly-cistronic RNA capable ofdirecting the translation of both the A and B subunits from the VT-1 andVT-2 genes, respectively.

[0266] In choosing a strategy for recombinant VT antigen production,there are three primary technical factors to consider. First, theappropriate VT antigen components representing the spectrum of toxinepitopes encountered in nature must be utilized.

[0267] Second, the protein antigens must be expressed at sufficientlevels and purity to enable immunization and large-scale antibodypurification. Third, the neutralizing epitopes must be preserved in theimmunogen and immunoabsorbant. Approaches that offer the greatestpromise for high level expression of periplasmically localized proteinswere developed. FIG. 10 shows the fusion constructs of VT components andaffinity tags.

[0268] A. Expression of affinity-tagged C-terminal constructs.

[0269] The VT1 and VT2 A and B subunits (SEQ ID NOS:1, 3, 5 and 7) arecloned into the pET-23b vector (Novagen). This vector is designed toallow expression of native proteins containing C-terminal poly-His tags.The vector utilizes a strong T7 polymerase promoter to drive high levelexpression of target proteins. The methionine initiation codon isengineered to contain a unique NdeI restriction enzyme site (CATATG).The VT1 and VT2 genes are engineered to convert the signal sequencemethionine codon into a NdeI site utilizing PCR mutagenesis. PCR primerswere designed which contain the sequence GCCAT fused to the first 20-24bases of the genes (starting at the ATG start codon of the signal tag;SEQ ID NOS:12-19, see Table below). Upon PCR amplification, the 5′ startcodon of each gene is converted to an NdeI site, compatible with thepET-23 vector-encoded NdeI site, allowing cloning of the amplified genesinto the vector without the addition of vector-encoded amino acids.

[0270] Primers containing the C-terminal 7 codons of each gene (21bases) fused to the sequence CTCGAGCC were synthesized, in order to adda C-terminal poly-His tag to each gene. The underlined bases are an XhoIsite, that is compatible with the XhoI site of the pET-23 vector. Theseprimers precisely delete the native stop codons, and when cloned intothe pET-23 vector, add a C-terminal extension of“LeuGluHisHisHisHisHisHis” (SEQ ID NO: 11). The following table liststhe primer pairs that are utilized to create PCR fragments containingthe A and B subunits derived from VT-1 and VT-2 toxin genes suitable forinsertion into the pET-23b vector. TABLE 9 Primers Toxin Gene andSubunit N-terminal Primer C-terminal Primer VT-1 Subunit A SEQ ID NO:12SEQ ID NO:13 VT-1 Subunit B SEQ ID NO:14 SEQ ID NO:15 VT-2 Subunit A SEQID NO:16 SEQ ID NO:17 VT-2 Subunit B SEQ ID NO:18 SEQ ID NO:19 VT-1Subunits A and B SEQ ID NO:12 SEQ ID NO:15 VT-2 Subunits A and B SEQ IDNO:16 SEQ ID NO:19

[0271] Thus, utilizing PCR amplification with the above modified N- andC-terminal primers, the A and B subunits of VT1 and VT2 are expressed asproteins containing an 8 amino acid C-terminal extension bearing anpoly-histidine affinity tag. The amino acid sequence of thehistidine-tagged VT-1 A subunit produced by expression from the pET-23bvector is listed in SEQ ID NO:21 (the associated DNA sequence is listedin SEQ ID NO:20); the amino acid sequence of the histidine-tagged VT-1 Bsubunit is listed in SEQ ID NO:23 (the associated DNA sequence is listedin SEQ ID NO:22); the amino acid sequence of the histidine-tagged VT-2 Asubunit is listed in SEQ ID NO:25 (the associated DNA sequence is listedin SEQ ID NO:24); the amino acid sequence of the histidine-tagged VT-2 Bsubunit is listed in SEQ ID NO:27 (the associated DNA sequence is listedin SEQ ID NO:26).

[0272] Both subunits may be expressed from a single expressionconstructs by utilizing SEQ ID NOS:12 and 15 to prime synthesis of theVT-1 toxin gene and SEQ ID NOS:16 and 19 to prime synthesis of the VT-2toxin gene. The resulting PCR products are cleaved with NdeI and XhoI,as described for the cloning of the subunit genes into the pET-23bvector. Expression of the A and B subunits from such an expressionvector, results in the expression of a native A subunit and a his-taggedB subunit. As the A and B subunits assemble into a complex, the presenceof the his-tag on only the B subunit is sufficient to allow purificationof the holotoxin on metal chelate columns as described below.

[0273] The proofreading Pfu polymerase (Stratagene) is utilized for PCRamplification to reduce the error rate during amplification. Genomic DNAfrom an E. coli O157:H7 strain is utilized as template DNA. Followingthe PCR, the amplification products are digested with NdeI and XhoI andcloned into the pCR-Script SK cloning vehicle (Stratagene) to permit DNAsequence analysis of the amplified products. The DNA sequence analysisis performed to ensure that no base changes are introduced duringamplification. Once the desired clones are identified by DNA sequencing,the inserts are then excised utilizing Ndel and XhoI, and cloned into asimilarly cut pET-23b vector to create the expression constructs.According to the published sequences, neither the VT1 nor VT2 genescontain either of these restriction sites.

[0274] The poly-His-tagged proteins produced by expression of the VT-1and VT-2 gene sequences in the pET-23b constructs are then purified byIMAC. This method uses metal-chelate affinity chromatography to purifynative or denatured proteins which have histidine tails (see e.g., K. J.Petty, “Metal-Chelate Affinity Chromatography,” in Current Protocols inMolecular Biology, Supplement 24, Unit 10.11B [1993]).

[0275] B. Expression of Toxin Containing N-terminal Affinity Tags

[0276] Two expression systems, pMa1-p2 and pFLAG-1 are utilized toattach an N-terminal affinity tag to the A subunits from the VT-1 andVT-2 toxins.

[0277] MBP-tagged constructs. To construct A chains containing themaltose binding protein (MBP) at the N-terminus of the A subunit, PCRamplified gene products are cloned into the pMa1-p2 vector (New EnglandBiolabs) as C-terminal fusions to a periplasmically-secreted version ofthe MBP. The MBP selectively binds to amylose resins and serves as anaffinity tag on the MBP/A subunit fusion protein. The pMa1-p2 vectorcontains an engineered factor Xa cleavage site, which permits theremoval of the affinity tag (ie., MBP) from the fusion protein afterpurification.

[0278] The MBP/A subunit fusions are generated as follows. The VT1 andVT2 A subunits are PCR-amplified utilizing the following DNA primers.SEQ ID NOS:28-31; SEQ ID NOS:28 and 29 comprise the 5′ and 3′ primers,respectively, for the amplification of the VT1 A subunit; SEQ ID NOS:30and 31 comprise the 5′ and 3′ primers, respectively, for theamplification of the VT2 A subunit. In both cases, the 5′ or N-terminalprimer contains the sequence CGGAATTC fused to the first codon of themature polypeptide (rather than the start of the signal peptide, sincethe MBP signal peptide is utilized). These 5′ primers contain anengineered EcoRI site that is not contained internally in either gene,that is compatible with the EcoRI site of the pMa1-p2 vector. The 3′ orC-terminal primers incorporate an XhoI site as described above for thegeneration of the His-tagged toxins, but in this case, the 3′ primer isdesigned to include the natural termination codon of the A subunits.

[0279] The genes are amplified, cloned into pCR-Script SK, and sequencedas described above. The inserts are then excised with EcoRI and AhoI,and cloned into EcoRI/Sall-cleaved pMa1-p2 vector (Sall and Xhol sitesare compatible). This construct allows expression and secretion of theVT1 and VT2 A subunit genes as C-terminal fusions with MBP. The aminoacid sequence of the MBPNVT-1A fusion protein is listed in SEQ ID NO:33(the associated DNA sequence is listed in SEQ ID NO:32). The amino acidsequence of the MBPNVT-2A fusion protein is listed in SEQ ID NO:35 (theassociated DNA sequence is listed in SEQ ID NO:34).

[0280] The resulting fusion proteins are then affinity purified on anamylose column and the bound fusion protein is eluted under mildconditions by competition with maltose. The MBP N-terminal-tagged Asubunits are cleaved with factor Xa and the MBP is removed bychromatography on an amylose column. The resulting A subunits whichcontain a 4 amino acid N-terminal extension are then used asirnmunogens.

[0281] Flag tag constructs. In an alternative embodiment, the VT1 andVT2 A subunit genes are engineered to contain the “flag tag” through theuse of the pFLAG-1 vector system. The flag tag is located between theOmpA secretion signal sequence and the authentic N-terminus of thetarget protein in the pFlag-1 vector. To construct N-terminalflag-tagged A chains, the EcoRllXhol A subunit PCR fragments (generatedas described above for the MBP fusion proteins) are cloned intoidentically cleaved pFlag-1 vector (Eastman-Kodak), to produce anexpression construct utilizing the OmpA signal peptide for secretion ofA subunit fusion proteins containing the flag peptide at the N-terminus.After secretion, the periplasmic protein contains the N-terminal 8 aminoacid flag tag, followed by 4 vector-encoded amino acids fused to therecombinant A subunit. The amino acid sequence of the flag tag/VT-1 Asubunit fusion protein is listed in SEQ ID NO:37 (the associated DNAsequence is listed in SEQ ID NO:36). The amino acid sequence of the flagtag/VT-2 A subunit fusion protein is listed in SEQ ID NO:39 (theassociated DNA sequence is listed in SEQ ID NO:38).

[0282] The flag tag fusion proteins are then purified by immunoaffinitychromatography utilizing a calcium-dependent monoclonal antibody(Antiflag M1; Eastman-Kodak). Mild elution of purified protein isachieved by chelating the calcium in the column buffer withethylenediamine tetraacetic acid (EDTA).

[0283] C. Evaluation of fusion construct expression.

[0284] The fusion constructs described above are expressed in E. colistrain BL21, or T7 polymerase-containing derivatives [e.g., BL21(DE3),BL21(DE3) pLysS, BL21(DE3)pLysE] (Novagen) for pET plasmids, andperiplasmically-secreted recombinant protein purified by affinitychromatography. Recombinant proteins are analyzed for correctconformation by testing the following parameters:

[0285] a) It is believed that the B subunit must associate intopentamers to be conformationally correct. This is assessed by reducingand native SDS-PAGE analyses of native and chemically-cross-linkedproteins and sizing HPLC;

[0286] b) It is believed that a properly folded A subunit is expected toretain its native enzymatic activity. This is tested by its capacity toinhibit protein synthesis in an in vitro toxicity assay;

[0287] c) It is believed that in vitro toxicity of assembled recombinantholotoxin can be assessed by comparison to commercially availableholotoxins to determine whether recombinant A and B subunits canassemble into functional holotoxin. The purified N-terminal-tagged Asubunits (after cleavage and purification from MBP or untreatedflag-tagged proteins) are combined in vitro with the corresponding Bchains, and their toxicity evaluated utilizing a quantitative microtitercytotoxicity assay, such as that described by M.K. Gentry and M.Dalrymple, “Quantitative Microtiter Cytotoxicity Assay for ShigellaToxin,” J. Clin. Microbiol., 12:361-366 (1980).

EXAMPLE 7 Verotoxin Clone Construction

[0288] In this Example, vectors expressing VT1 A and B, and VT2A and Bsubunits with a C-terminal his-tag were constructed, as well as vectorsexpressing VT1A and VT2A as a fusion with the MBP. In addition, vectorscapable of expressing the native VT1 A and VT2A subunits (i.e., withoutan affinity tag) were also generated. Table 10 provides a summary of VTconstructs and provides information concerning the parent vector, theaffinity tag (if present) and the antibiotic selection employed forgrowth of the plasmid construct. In Table 10, the term “L+” indicatesthat the expression vector encodes the preprotein form of the verotoxinsubunit (i.e., the plasmid utilizes the naturally occurring signalsequence for secretion of the verotoxin subunit into the periplasm ofthe host cell). “L-” indicates that the expression vector encodes themature form of the verotoxin subunit (i.e., sequences encoding thenaturally occuring signal sequence of the verotoxin subunit are notpresent and therefore the protein will remain intracellular).

[0289] The predicted amino acid sequences of the subunit proteinsexpressed by the plasmids listed in Table 10 are as follows:pET23hisVTIA L+and pET24hisVT1 A L+(SEQ ID NO:21); pET23hisVT1 A L-(amino acid residues 23-323 of SEQ ID NO:21); pET23hisVT2A L+andpET24hisVT2A L+(SEQ ID NO:25); pET23hisVT2A L- (amino acid residues23-326 of SEQ ID NO:25); pET23hisVT1 B L+and pET24hisVT1 B (SEQ IDNO:23); pEThisVT1 B L- (amino acid residues 20-97 of SEQ ID NO:23);pET24VTIB and pET24T7VT1 B (SEQ ID NO:3); pET23hisVT2B L+ andpET24hisVT2B L+ (SEQ ID NO:27); pET24VT2B, pET24T7VT2B and pET24T7VT2BlacIq- (SEQ ID NO:8); pET24hisVT2B L- (amino acid residues 20-97 of SEQID NO:27); pMa1VT1 A (SEQ ID NO:47; the nucleotide sequence encoding theMBPNVT1A fusion protein is provided in SEQ ID NO:46); pMa1VT2 A (SEQ IDNO:49; the nucleotide sequence encoding the MBPNT2A fusion protein isprovided in SEQ ID NO:48). TABLE 10 Plasmid Constructs Transcrip- VTParent tional Affinity Se- Plasmid Subunit Vector Control Tag lectionpET23hisVT1 A VT1A(L+) pET23 T7 6X HIS Amp L+ pET24hisVT1 A VT1A(L+)pET24 T7lac 6X HIS Kan L+ pET23hisVT1 A VT1A(L−) pE23 T7 6X HIS Amp L−pET23hisVT2 A VT2A(L+) pET23 T7 6X HIS Amp L+ pET24hisVT2 A VT2A(L+)pET24 T7lac 6X HIS Kan L+ pET23hisVT2 A VT2A(L−) pET23 T7 6X HIS Amp L+pET23hisVT1 B VT1B(L+) pET23 T7 6X HIS Amp L+ pET24hisVT1 B VT1B(L+)pET24 T7lac 6X HIS Kan L+ pET23hisVT1 B VT1B(L−) pET23 T7 6X HIS Amp L−pET24VT1 B VT1B(L+) pET24 T7lac NONE Kan L+ pET24T7VT1 B VT1B(L+)pET24VT T7 NONE Kan 1B pET23hisVT2 B VT2B(L+) pET23 T7 6X HIS Amp L+pET24hisVT2 B VT2B(L+) pET24 T7lac 6X HIS Kan L+ pET24VT2 B VT2B(L+)pET24 T7lac NONE Kan pET24T7VT2 B VT2B(L+) pET24VT T7 NONE Kan 2BpET24T7VT2 B VT2B(L+) pET24VT T7lac NONE Kan lacIq- 2B pET24hisVT2 BVT2B(L−) pET24 T7lac 6X HIS Kan L− pMalVT1 A VT1A(L−) pMAL-p2 ptac MPBAmp pMalVT2 A VT2A(L−) pMAL-p2 ptac MPB Amp pMalVT2 A VT2A(L−) pMalVT2ptac MPB Amp (BamHI) A

[0290] A. His-Tagged Constructs

[0291] The pET vectors (Novagen, Madison, Wisc.) were used to producehis-tagged recombinant subunits. These vectors were designed to expresseach subunit with a C terminal 6X his-tag (i.e., a tag comprised of sixhistidine residues) to facilitate affinity purification usingimmobilized metal chelate columns. The coding regions for each subunitwere cloned using PCR amplification from E. coli 933 genomic DNA. Thesubunits were amplified from genomic DNA using the L+ and L- primers. L+indicates a 5′ primer that contains the native periplasmic secretionsignal, L- indicates a 5′ primer that is designed to delete the nativesecretion signal and produces the recombinant protein intracellularly.Table 11 lists the primers used to make the his-tagged constructs. TABLE11 Primers Used for Amplification of Genomic E. coli 933 DNA Subunit SEQID Primer Primer Sequence NO: VT1A 5′ GCCATATGAAAATAATTATTTTTAGAGTG SEQID L+ (NdeI site underlined) NO: 12 VT1 A 5′ GCCATATGAAGGAATTTACCTTAGACSEQ ID L− (NdeI site underlined) NO: 40 VT1 A 3′GGCTCGAGACTGCTAATAGTTCTGCGCAT SEQ ID (XhoI site underlined) NO: 13 VT2 A5′ GCCATATGAAGTGTATATTATTTAAATGG SEQ ID L+ (NdeI site underlined) NO: 16VT2 A 5′ GCCATATGCGGGAGTTTACGATAGAC SEQ ID L− (NdeI site underlined) NO:41 VT2 A 3′ GGCTCGAGTTTACCCGTTGTATATAAAAAC SEQ ID (XhoI site underlined)NO: 17 VT1 B 5′ GCCATATGAAAAAAACATTATTAATAGC SEQ ID L+ (NdeI siteunderlined) NO: 14 VT1 B 5′ GCCATATGACGCCTGATTGTGTAACT SEQ ID L− (NdeIsite underlined) NO: 42 VT1 B 3′ GGCTCGAGACGAAAAATAACTTCGCTGAA SEQ ID(XhoI site underlined) NO: 15 VT2 B 5′ CGCATATGAAGAAGATGTTTATGGCG SEQ IDL+ (NdeI site underlined) NO: 18 VT2 B 5′ GCCATATGGCGGATTGTGCTAAAGG SEQID L− (NdeI site underlined), NO: 43 VT2 B 3′GGCTCGAGGTCATTATTAAACTGCACTTC SEQ ID (XhoI site underlined) NO: 19

[0292] The E. coli O157:H7 933 strain was obtained from Dr. O'Brien(See, Example 5), and grown in L broth (Maniatis et al). Highmolecular-weight E. coli genomic DNA was isolated essentially asdescribed by Wren and Tabaqchali, “Restriction endonuclease DNA analysisof Clostridium difficile,” J. Clin. Microbiol., 25:2402-2404 [1987]),with the exceptions being that proteinase K and sodium dodecyl sulfate(SDS) were used to disrupt the bacteria, and methods for CTABprecipitation as described in another reference (Ausubel et al., (eds.),in Current Protocols in Molecular Biology pages 2.4.1-2.4.2 [1995]) wereused to remove carbohydrates from the cleared lysate. The integrity andyield of genomic DNA was assessed by comparison with a serial dilutionof uncut lambda DNA after electrophoresis on an agarose gel.

[0293] The gene fragments were cloned by PCR, utilizing a proofreadingthermostable DNA polymerase (native Pfu polymerase; Stratagene). Thispolymerase was chosen as its high fidelity of this polymerase reducesthe mutation problems associated with amplification by error pronepolymerases (e.g., Taq polymerase). PCR amplification was performedusing the indicated PCR primer pairs with two amplifications conductedfor each subunit (ie., 5′ L+primer/3′ primer and 5′ L- primer/ 3′primer) in 50 μl reactions containing 10 mM Tris-HCl (pH 8.3), 50 mMKCl, 1.5 MM MgCl₂, 200 μM of each dNTP, 0.2 μM of each primer, and 50 ngE. coli genomic DNA. Reactions were overlayed with 100 μl mineral oil,heated to 94° C. 4 min, 0.5 μl native Pfu polymerase (Stratagene) wereadded, and the reaction cycled for thirty times (94° C. for 1 min, 50°C. for 2 min, 72° C. for 4 min), followed by 10 min at 72° C.). Then, 101 μl aliquots of amplified DNA were resolved on agarose gels, andamplified DNA gel purified using the Prep-A-Gene kit (Biorad), andligated to pCRScript vector DNA (Stratagene). Recombinant clones wereisolated and confirmed by restriction digestion, or sequencing (VT1B andVT2B clones) using standard recombinant molecular biology techniques(Sambrook et al., 1989).

[0294] Expression plasmids were constructed as follows, andmanipulations were identically performed for L+and L- clones. Thesubunit clones in the PCRscript vector were cleaved with NdeI/XhoI, andgel purified subunit fragments were cloned into gel purifiedNdeI/XhoI-digested pET23b vector. The resulting clones, designated aspET23hisVT l AL+, pET23hisVT1 AL−, pET23hisVT 1 BL+, pET23hisVT 1 BL−,pET23hisVT2AL+, pET23hisVT2AL−, and pET23hisVT2BL+, were ampicillinresistant and expressed the subunits utilizing the T7 promoter. Allclones were confirmed by restriction mapping.

[0295] In addition, T71ac, kan, Laclq L+ or L− clones were alsoconstructed as described above, substituting NdeI/XhoI-cleaved pET24vector for pET23 vector. These 5 clones, designated as pET24hisVT1AL+,pET24hisVT1 BL+, pET24VT2AL+, pET24VT2BL+, and pET24VT2BL−, wereconfirmed by restriction digestion.

[0296] B. Construction of Vectors Without Affinity Tags

[0297] Vectors lacking affinity tags were also produced. These vectorswere designed to express the VT1B and VT2B subunits periplasmically,without any additional amino acids. The subunits are contemplated tohave a native sequence, since the coding region of the expressionconstructs were unaltered from the original genes. The subunits werePCR-amplified from E. coli O157:H7 933 genomic DNA, the amplified bandswere gel purified and then cloned into the pCRScript vector as describedabove. The amplifications were performed utilizing the VTIB or VT2B 5′L+primers described above (i.e., SEQ ID NO:14 or 18), and the following3′ primers. The VT1B native 3′ primer had the sequence5′-GGCTCGAGTCAACGAAAAATAACTTCGCTGAA-3′ (XhoI site underlined) (SEQ IDNO:44); and the VT2 B native 3′ primer had the sequence5′-GGCTCGAGTCAGTCATTATTAAACTGCACTTC-3′ (XhoI site underlined) (SEQ IDNO:45).

[0298] The initial expression constructs were constructed by cloning theNdeIIXhoI fragments from the pCRScript clones into NdeI/XhoI-cleavedpET24a vector. The clones were designated pET24VT1 B or pET24VT2B, andwere confirmed by complete sequencing of the inserts. These clonescontained the lacIq gene, and verotoxin subunits expression was drivenby the T71ac promoter. To increase expression yields, pET24-derivedplasmids in which the T7 promoter was substituted for the T71ac promoterwere constructed. The insert containing XbaI/XhoI fragments of pET24VT1B and pET24VT2B were cloned into XbaI/XhoI released vector from thepHisBotE kan laclq T7 vector (described in co-pending U.S. patentapplication Ser. No. 08/704,159, herein incorporated by reference). Aequivalent vector backbone can be generated as follows. pET24 isdigested with XbaI and SapI and the ˜2.6 kb band containing the kan^(R)gene, fl origin and plasmid origin is isolated and ligated to the 996 bpXbaI/SapI fragment from pET23. The resulting plasmid contains the T7promoter but lacks the lacIq gene. The resulting plasmid is thendigested with BglII and SapI and the large fragment is isolated andligated to the ˜2.7 BglII/SapI fragment from pET24. The final constructcontains the kan^(R) gene, the T7 promoter and the lacIq gene. Theresultant clones, pET24T7VTIB and pET24T7VT2B, were confirmed bysequencing. Finally, a VT2B expressing construct that was kanamycinresistant, contained the T71ac promoter but had deleted the laciq genewas constructed, by insertion of the insert containing XhoI/BglIIfragment from pET24VT2B into the XhoI/BglII vector backbone releasedfrom the pHisBotA kan plasmid (described in co-pending U.S. patentapplication Ser. No. 08/704,159, supra). An equivalent vector backbonecan be generated as follows. pET24 is digested with BglII and SapI andthe ˜2.6 kb band containing the T71ac promoter, kan^(R) gene, fl originand the plasmid origin is isolated. pET23 is digested with BglII andSapI and the 996 bp fragment is isolated and ligated to the ˜2.6 kbfragment form pET24. The resulting plasmid contains the kan^(R) gene andthe T71ac promoter but lacks the lacIq gene (referred to as a lacIq-derivative of pET24).

[0299] C. MBP Fusions

[0300] In this experiment, alternative methods for tagging the proteinswere used with the pMa1vector/expression system (New England Biolabs).These vectors were designed to express the recombinant subunits withmaltose binding protein (30 kd), in order to allow affinity purificationof the recombinant subunits on amylose resins. In this experiment,pMa1VT1 A and pMa1VT2A plasmids were constructed.

[0301] For pMa1VT1 A, the insert from a pCRScript L+ clone containingthe amplified VT1A gene in the appropriate orientation was excised withBamHI/XhoI and cloned into BamHI/SalI-cleaved pMAL-c2 (New EnglandBiolabs). The resultant clone was confirmed by restriction digestion.

[0302] For pMa1VT2A, the insert from a pCRScript L+ clone containing theamplified VT2A gene in the appropriate orientation was excised withBamHI/XhoI and cloned into BamHI/SalI-cleaved pMAL-p2. The resultantclone was confirmed by restriction digestion. As discussed below, theVT2A PCR amplification product in this pCRScript L+ clone appeared tohave deleted a nucleotide at the 5′ end that resulted in a shift (-1) inthe reading frame of the VT2A subunit. Therefore, a derivative clonepMa1VT2A(BamHI) was also constructed that inserted 4 bp and induced a+1frameshift relative to the parent vector by digesting pMa1VT2A withBamHI, filling in the BamHI site using the Klenow enxzyme and all 4dNTPs followed by circularization of the plasmid. The nucleotidesequence encoding the MBPJVT2A subunit expressed by pMa1VT2 A(BamHI) islisted in SEQ ID NO:X; the amino acid sequence of this fusion protein isprovided in SEQ ID NO:Y.

EXAMPLE 8 EXPRESSION OF VEROTOXIN HIS-TAGGED CLONES

[0303] In this Example, methods used for the expression of his-taggedclones was developed. In this Example, the pET vector derived verotoxinexpression constructs were transformed into BL21(DE3) containing E. colicell lines for expression. However, several expression constructs werefound to be toxic in these cell lines. The viability of the various celllines is summarized in the following Table. TABLE 12 Growth andViability of Verotoxin-Expression Constructs in E. coli Cell Lines.Plasmid BL21 (DE3) BL21(DE3)plysS BL21(DE3)plysE pET23hisVT1AL +Negative Positive Positive pET23hisVT2AL + Negative Positive PositivepET23hisVT1BL + Negative Positive Positive pET23hisVT2BL + NA NegativePositive pET23hisVT1AL − Positive Positive NA pET23hisVT2AL − NegativeSlow growth NA pET23hisVT1BL − Negative Slow growth NA pET24hisVT2BL −Positive NA NA pBT24hisVT1AL + Positive NA NA pET24hisVT2AL + PositiveNA NA pET24hisVT1BL + Positive Positive NA pET24hisVT2BL + PositivePositive NA pET24VT1B Positive NA NA pET24T7VT1B Positive NA NApET24VT2B Positive NA NA pET24T7VT2B Negative NA NA pET24VT2BlacIq-Negative NA NA

[0304] From these results, it appeared that expression of any of theverotoxin subunits is toxic to E. col, and that the only clones that areviable in the BL21(DE3) cell line are those in which subunit expressionis tightly repressed in uninduced cells, either by use of a T71acpromoter or the lacIq gene or both.

[0305] Protein expression was induced in 1 liter shaker flask culturesutilizing recombinant plasmids in the BL21(DE3)-derived E. coli strains.Procedures for protein induction, SDS-PAGE, and Western blot analysiswere described in detail in Williams et al (Williams et al., in Gloverand Hames (eds.), DNA4 Cloning 2: Expression Systems, A PracticalApproach, 2d ed., IRL Press [1995], pages 15-57).

[0306] In brief, 1 liter 2XYT +0.2% glucose +either 40 jig/ml kanamycin(pET24 plasmids) or 100 tg/ml ampicillin (pET23 plasmids) cultures ofbacteria were induced to express recombinant protein by addition of IPTGto lmM. For optimal results, cultures were grown at 30-32° C., overnightand induced when the cell density reached >2 OD₆₀₀. Induced protein wasallowed to accumulate for 2-4 hrs after induction. Induction at lowerOD₆₀₀ readings (e.g., 0.5-1.0) resulted in the accumulation of loweroverall levels of verotoxin subunits, since induction of subunitexpression halted E. coli cell growth and final cell pellets weretherefore dramatically smaller. In the case of VT1B and VT2B expressingconstructs, the verotoxin subunits were insoluble if grown and inducedat 37° C.

[0307] The cells were harvested by centrifugation 10 min at 5000 rpm ina JALO rotor at 4° C. The pellets were resuspended in a total volume of40 mls IX binding buffer (5 mM imidazole, 0.5 M NaCl, 50 mM NaPO₄, pH8.0) +1 mg/ml lysozyme (if lysS or lysE plasmid was not present),transferred to two 50 ml Oakridge tubes and frozen at −70° C, for atleast 1 hr. The tubes were thawed and the cells lysed by sonication (4 X20 second bursts) on ice. The suspension was clarified by centrifugation20-30 min at 9,000 rpm (10,000 xg) in a JA-17 rotor. The soluble lysatewas batch-absorbed to 7 mls of a 1:1 slurry of NiNTA resin: bindingbuffer by stirring 2-4 hr at 4° C. The slurry was centrifuged 1 min at500 xg in a 50 ml tube (Falcon), resuspended in 5 mls binding buffer andpoured into a 2.5 cm diameter column (Biorad). The column was attachedto an UV monitor (Isco), and washed with binding buffer, then bindingbuffer +20-40 mM imidazole until baseline was established. Bound proteinwas eluted in elution buffer (50 mM NaPO₄, 0.3 M NaCl, 20% glycerol, 250mM imidazole, pH 8.0) and stored at +4 or −20° C.

[0308] Samples of total, soluble, flow through and eluted protein werethen resolved by SDS-PAGE. Protein samples were prepared by mixing 1 pItotal (T), soluble (S) or flow through (A) protein with 4 RI PBS and 5Rl 2X SDS-PAGE sample buffer, or 5 μl eluted (E) protein and 5 pI 2XSDS-PAGE sample buffer (See, co-pending U.S. patent application Ser. No.08/704,159). The samples were heated to 95° C. for 5 min, cooled, and 5or 10 μls were loaded on 12.5% SDS-PAGE gels. Broad range molecularweight protein markers (BioRad) were also loaded to allow the MW of theidentified fusion proteins to be estimated. After electrophoresis,protein was detected either generally by staining gels with Coomassieblue. A representative Coomassie stained 20% SDS-PAGE gel demonstratingpurification of his-tagged VT1B and VT2B proteins is shown in FIG. 11.

[0309] In FIG. 11, lanes 1-3 are from a pET24hisVT1 BL+BL2I(DE3)purification, lanes 4-7 are from a pET24hisVT2BL− BL21(DE3)purification, and lane 8 is from a pET24hisVT2BL+BL21(DE3). Lanes 1 and4 are total protein, lanes 2 and 5 are soluble protein, lane 6 isprotein after NiNTA absorption (5× concentration), lanes 3, 7, and 8 areNiNTA column elution samples, and lane 9 contains broad range molecularweight markers (BioRad). As shown, the VT1B protein is smaller than theVT2B protein. The fact that the VT2BL+ and VT2BL− proteins areidentically sized indicated that the VT2BL+ protein has the leaderpeptide cleaved off and is thus likely to be periplasmically secreted.

[0310] Purification yields and purity from 11 shaker flask cultures, forseveral his-tagged plasmid/host cell combinations, are shown in Table 13(purity was estimated by visual inspection of the Coomassie-stainedSDS-PAGE gels). In Table 13, the protein concentration was estimatedusing 1 mg/ml solution =2 OD₂₈₀/ml. TABLE 13 Purification of His-TaggedVerotoxin Proteins Wash buffer Yield& Plasmid Host strain imidazoleconc. Purity pET24hisVT1BL + BL21(DE3) 40 mM imidazole 2.5-5 mg/l >50%purity pET24hisVT2BL + BL21(DE3) 40 mM imidazole 60-70 mg/l >90% puritypET24hisVT1AL + BL21(DE3) 20 mM, then <1 mg/l 40 mM imidazole <10%purity pET24hisVT2AL + BL21(DE3) 20 mM, then <1 mg/l 40 mM imidazole<10% purity pET23hisVT1BL + BL21(DE3)plysS 40 mM imidazole 2.5-5mg/l >50% purity pET23hisVT2BL + BL21(DE3)plysE 40 mM imidazole >30mg/l >90% purity pET23hisVT1AL + BL21(DE3)plysS 20 mM imidazole <1 mg/l<10% purity pET23hisVT2AL + BL21(DE3)plysS 20 mM imidazole <1 mg/l <10%purity pET23hisVT1AL − BL21(DE3) 20 mM, then <1 mg/l 40 mM imidazole<10% purity pET23hisVT2AL − BL21(DE3)plysS 20 mM, then <1 mg/l 40 mMimidazole <10% purity pET23hisVT1BL − BL21(DE3)plysE 20 mM, then <1 mg/l40 mM imidazole <10% purity pET24hisVT2BL − BL21(DE3) 20 mM, then >40mg/l 40 mM imidazole >90% purity

[0311] Expression and purification of large quantities (>40 mg/1) of theVT2B subunit was obtainable utilizing any of the described expressionsystems. However, due to toxicity of the VT2B subunit, strict uninducedpromoter control was necessary to allow cell viability. This wasaccomplished by coexpression with the plysE plasmid in the case of thepET23hisVT2BL+ plasmid, or the presence of the lacIq gene and the T71acpromoter in the pET24hisVT2BL+ and L− plasmids. Due to the need fordisulfide bond formation and pentamer assembly, the vectors that allowedperiplasmic secretion of the protein (L+) were found to be preferable,since intracellular E. coli is a reducing environment. In addition, dueto scaleup and plasmid stability concerns, the pET24 construct was foundto be preferable to the pET 23 construct.

[0312] Expression and purification of moderate quantities (5 mg/l) ofthe VT1B subunit was also attainable utilizing any of the describedexpression systems. The VT1B subunit is less toxic than the VT2Bsubunit, allowing less stringent control of uninduced verotoxinexpression. Due to the need for disulfide bond formation and pentamerassembly, the vectors that allow periplasmic secretion of the protein(L+) were found to be preferable. As with the VT2B subunit, due toscaleup and plasmid stability concerns, the pET24 construct was found tobe preferable to the pET 23 construct.

[0313] In contrast to the VT1B yields, very poor yields of purified VT1Aor VT2A subunits were obtained, utilizing either L+ or L− vectors.

EXAMPLE 9 VT1 Subunit A and VT2 Subunit A MBP Clone Expression

[0314] Due to the poor recovery of his-tagged VT1A and VT2A protein,expression of MBP fused VT1 A and VT2A subunits was undertaken in thisExample. Large scale (1 liter) cultures of pMa1VT1 A, pMa1VT2A andpMaIVT2A(BamHI) in the BL21 plysS strain were grown, induced, andsoluble protein fractions were isolated. One liter cultures were grownat 30° C. in 2XYT broth containing 0.2% glucose and 100 μg/mlampicillin. Recombinant protein expression was induced by addition ofIPTG to 1.0 mM at approximately 1.0 OD₆₀₀ cell density. Cultures wereinduced for 2-3 hrs. The cells were collected by centrifugation in aJALOrotor (Beckman) at 5000 rpm for 10 min. at 4° C. The cell pelet wasresuspended in 40 ml PBS and frozen at −70° C. The samples were thawedin warm water and sonicated using a Branson Sonifier with the microtip(20 sec/pulse, 8 pulses total). The sonicated material was then andclarifed by centrifugation (Beckman JS13 rotor at 10,000 rpm at 4° C.for 20 min.). The supernatant (i.e., the soluble extract) was thendecanted. Williams et al 1995, supra.

[0315] The soluble extracts were diluted to 200 mls with PBS,chromatographed over an amylose resin (New England Biolabs) columrn andthe flow through material (i.e., proteins that did not bind to theresin) was collected. The column was then washed with PBS until a stablebaseline was established, and eluted with PBS containing 10 mM maltoseas described (Williams et al. [1995], supra). Protein yields were 22 mg(pMa1VTIA), 13.5 mg (pMaIVT2A) or 12.5 mg [pMa1VT2A(BamHI)] from 1 literstarting volume for each recombinant (protein concentration estimatedusing 1 mg/ml 2 OD₂₈₀/ml). Coomassie stained 10% SDS-PAGE gels ofsamples of these purifications are shown in FIGS. 12 and 13.

[0316] In FIG. 12, lanes 2-5 contained and lanes 6-9 are pMa1VT2Aprotein preparations. Lanes 2 and 6 contained total protein, lanes 3 and7 contained soluble protein, lanes 4 and 8 contained flow throughprotein, lanes 5 and 9 contained eluted protein samples, and lane 1contained broad range molecular weight markers (Biorad).

[0317] In FIG. 13, lanes 2-5 contained pMa1VT2A(BamHI) proteinpreparations. Lane 2 contained total protein, lane 3 contained solubleprotein, lane 4 contained flow through protein, lane 5 contained elutedprotein samples, and lane 1 contained broad range molecular weightmarkers (Biorad). As shown FIGS. 12 and 13, significant amounts ofpredicted full length VT1A and VT2A proteins (arrows) were produced bythe pMa1VT1A and pMa1VT2A(BamHI) plasmids, but not the pMa1VT2A plasmid.

[0318] Although it is not necessary to the practice of the presentinvention, it was assumed that the VT2A PCR amplification productcontained within the pCRScript L+ clone containing the VT2A gene haddeleted a nucleotide at the 5′ end, as introduction of a frameshift bythe addition of 4 bases in the filled BamHI site of the pMa1VT2B(BamHI)vector resulted in accumulation of the predicted full length protein.The identity of the predicted full length VT1A and VT2A proteins wasconfirmed by Western blot analysis. Verotoxin protein was identifiedutilizing a chicken anti-VT1 IgY.

[0319] For Westerns, samples of the pMa1VT1 A, pMa1VT2A andpMa1VT2A(BamHI) elutions were resolved on SDS-PAGE gels as describedabove, the gels were blotted, and protein transfer confirmed by PonceauS staining (See, Williams et al., [1995] supra). After blocking theblots for 1 hr at room temp in PBS + 0.1% Tween-20 (PBST) containing 5%milk (Blocking Buffer), 10 ml of a 1/1000 dilution of a anti-VTIholotoxin IgY PEG preparation in Blocking Buffer was added and the blotswere incubated a further 1 hr at room temperature. The blots were washedand developed with alkaline phosphatase, using a rabbit anti-chickenalkaline phosphatase conjugate as the secondary antibody (See, Williamset al., [1995], supra). This analysis confirmed that the full lengthproteins detected by Coomassie gel analysis in FIGS. 12 and 13 wereimmunoreactive with the anti-VT1 antibody preparation. The reactivity ofthe VT2A protein with the VT1 antiserum was predicted, as the VT1antiserum was demonstrated to cross-react with the VT2 protein inprevious Examples. From Coomassie gel staining, it was estimated that50% of the pMa1VT1 A elution and 10% of the pMa1VT2A(BamHI) elution wasfull-length fusion protein. This corresponds to 11 mg/l (VT1A) or 1.25mg/l (VT2A) yields of full length verotoxin subunit using theseexpression systems.

EXAMPLE 10 Expression of Native Verotoxin B Subunit

[0320] In this Example, the pET24VT1 B (T7 and T71ac) and pET24VT2Bexpression vectors were evaluated for their utility in expression ofnative VT1B and VT2B subunits. The pET24VT2B plasmid was selected forstudy, since this is the only VT2B expression vector that is viable inthe BL21(DE3) cell line (see Table 12). Expression levels from pET24VT1B and pET24T7VT1 B were evaluated by Western Blot analysis of total andsoluble protein extracts from small scale culture.

[0321] A. Expression of VT1 B

[0322] Fifty ml 2XYT+40 ag/ml kanamycin cultures of each plasmid in theBL21(DE3) strain were grown until OD₆₀₀>2.0, and verotoxin expressionwas induced for 3 hrs after addition of IPTG to lmM. A total of 10 OD₆₀₀units of cells (e.g., 5 mls of cells at OD₆₀₀=2/ml) were removed beforeand after induction and pelleted 5 min at maximum rpm in a benchtopcentrifuge. The pellets were resuspended in 1 ml of 50 mM NaHPO₄, 0.5 MNaCl, 40 mM imidazole buffer, pH 6.8, containing 1 mg/ml lysozyme. Thesamples were incubated 20 min. at room temp and stored overnight at −70°C. Samples were thawed completely at room temperature and sonicated 2×10seconds with a Branson sonifier 450 microtip probe at # 3 power setting.The samples were centrifuged 5 min at maximum rpm in a microfuge. Twentylil protein samples were removed to 20 μl 2× sample buffer, before orafter centrifugation, for total and soluble protein extractsrespectively. The samples were heated to 95° C. for 5 min, cooled and 5(15 lane gels) or 10 (10 lane gels) μls loaded on 20% SDS-PAGE gels.High molecular weight protein markers (Biorad) were also loaded, toallow estimation of the molecular weight of identified fusion proteins.After electrophoresis, VT1 B subunit protein was detected specifically,by blotting to nitrocellulose for Western blot detection utilizing aVT1B reactive monoclonal antibody. Western blot analysis was performedas described in Example ________, utilizing 1 μg/ml SLT13C4 (anti-VT1 Bmonoclonal; Toxin Technology, Sarasota, Fla.) as primary, and 1/1000diluted anti-mouse alkaline phosphatase conjugate (Kirkegaard PerryLaboratories, Gaitherburg, Md.) as secondary antibodies. Noimmunoreactive protein was detected in uninduced cell extracts fromeither cell line, while a single immunoreactive band of the predictedmolecular weight was detected in the induced cell extract from both celllines. The induced verotoxin expression level was much higher with thepET24T7VT1 B expression construct; this construct was selected forfurther study. This analysis demonstrated that both expression systemsproduce inducible verotoxin subunit proteins.

[0323] B. Collection of VT1B and VT2B Total, Soluble, Periplasmic, andCulture Broth

[0324] Cultures of VT1B and VT2B were grown and induced, and total,soluble, periplasmic and culture broth samples collected, to allowprotein quantification and subcellular localization of expressedverotoxins to be determined. One liter 2XYT+0.2% glucose +40 μg/mlkanamycin cultures of pET24VT2B and pET24T7VT1 B were grown at 30-32°C., until OD₆₀₀, =1-2, and verotoxin expression induced by addition ofIPTG to lmM, and the cultures grown 2.5-3 hrs. Total and solubleextracts were prepared from 10 OD₆₀₀ units of cells as described above.Samples of clarified culture broth were retained for detection ofsecreted verotoxin subunit. The 1 liter cultures were pelleted bycentrifugation 10 min at 8000×g. Osmotic shock and PMB-inducedperiplasmic extracts were then prepared. Half of the pellet wasresuspended in 200 ml 30 mM Tris, 20% sucrose, pH 8.0 and used toprepare an osmotic shock solution exactly as described in CurrentProtocols in Molecular Biology (Current Protocols, 16.6.7 alternativeprotocol). PMB extraction was performed on the other half of thepelleted cultures by first washing with PBS, resuspending them in 80 mlPBS, with 3.2 ml 50 mg/ml PMB (Sigma) added, and the solution wasincubated 10 min on ice. The PMB extracted cells were then centrifuged20 min at 11,000×g. The supernatant compriseds the PMB extractedperiplasmic extract.

[0325] C. ELISA Quantification of VT1 B and VT2 B Subunits

[0326] Expression levels of VT1B or VT2B subunits in each of theextracts prepared above were quantified utilizing a quantitative ELISAprotocol. In this procedure, 96-well microtiter plates (Falcon, Pro-BindAssay Plates) were coated by placing 100 μl volumes of either mouseanti-VT1 B (SLT 13C4 monoclonal; Toxin Technology) or mouse anti-VT2B(SLT 2B12 monoclonal; Toxin Technology) at 10 μg/ml in 100 mM sodiumbicarbonate, pH 9.0, in each well and incubating overnight at roomtemperature. The next morning, the coating suspensions were decanted,and 100 μl of 1.0% gelatin (Sigma) in PBS (blocking solution) was thenadded to each well, and the plates were incubated for 1 hr. at roomtemperature. The blocking solution was decanted, the wells washed 2×with PBS+0.1% Tween-20, and duplicate samples of 100 μl of sample addedto the first well of a dilution series, and 100 μl of ⅕ diluted samplewere added to each subsequent dilution well. To produce a standardcurve, 100 μl of a 2 fold serial dilution from 1-50 ng/ml of purifiedVT1B or VT2B subunit (his-tagged subunit material purified in asdescribed in Example ______, with 2 OD₂₈₀/ml estimated to be 1 mg/ml)was added in duplicate to dilution wells. All dilutions were in sampledilution buffer (PBS+ 0.1% Tween-80, 0.1% gelatin, 0.5% BSA, 20%glycerol, 0.05% NaAzide). The plates were incubated 1 hr at roomtemperature. The protein solutions were decanted and the plates werewashed 4× with PBS+0.5% Tween-20. Next, 100 μl/well of either 1/1000diluted chicken anti-VT1 B 4× PEG preparation (for VT1B ELISA) or 1.5jig/ml affinity purified chicken anti-VT2B (for VT2B ELISA) diluted inconjugate dilution buffer (PBS+ 0.1% Triton X100, 0.2% gelatin and 0.05%NaAzide) was added. After 30 min incubation at room temperature, thewells were washed 4× with PBS+0.5% Tween-20, and 100 μl/well of 1/1000diluted anti-chicken alkaline phosphatase conjugate (Sigma) in conjugatedilution buffer was added. After ½ hr at room temperature, the wellswere washed 2× PBS +0.5% Tween-20, then 2× PBS.

[0327] The plates were developed by the addition of 100 μl of a solutioncontaining 1 mg/ml para-nitro phenyl phosphate (Sigma) dissolved in 50mM Na₂CO₃, 10 mM MgCl₂ (pH 9.5), to each well, and incubating the platesat room temperature in the dark for 5-30 min. The absorbency of eachwell was measured at 410 nm using a plate reader (Dynatech MR 700). Theconcentration of verotoxin subunit in each unknown sample was estimatedby comparison with the absorbance of the reference standard solution (atan antigen concentration in which the reference standard absorbanceincreased 2-fold with a 2-fold increase in concentration). The ELISAassays were specific to the relevant subunits, since 0 μg/mlconcentrations were obtained when VT2B soluble lysates were read in theVT1B assay, or when VT1B soluble lysates are read in the VT2B assay. Theestimated concentrations of the subunits for each expression system areshown in Table 14. In this Table, the concentrations listed are eitherVT1B (for VT1B row) or VT2B (for VT2B row) verotoxin protein. TABLE 14Subcellular Localization and Concentration of Verotoxin SubunitExpression Total Soluble Periplasm Periplasm Culture Plasmid cellularcellular (Osmotic) (PMB) Soup pET24T7VT1B 0.8-1.5 mg/l 0.8-1.5 mg/l 1.2mg/l 0.14 mg/l 0.16 mg/l pET24VT2B 3.0-7.5 mg/l 3.0-7.5 mg/l 1.2 mg/l0.7-1.75  2.5 mg/l mg/l

[0328] This analysis demonstrated that the bulk of the expressed VT1 Bsubunit was periplasmically located, and quantitatively released byosmotic shock but not PMB extraction. The VT2B subunit is both cellassociated (of which approximately 1/2 is periplasmically localized) andsecreted into the culture supernatant. Thus, successful purificationstrategies for isolation of these subunits were periplasmic proteinpreparations for VT1B, and either periplasmic, whole cell, or culturebroth protein preparations for VT2B.

EXAMPLE 11 FERMENTATION CULTURE OF THE pET24VT2B BL21(DE3) CELL LINE

[0329] In this Example, large scale purification of the VT2B proteinusing a 10 liter fermentation of BL21(DE3) cells containing thepET24VT2B plasmid was performed.

[0330] The pET24VT2B plasmid was transformed into the BL21(DE3) strain,and glycerol stocks prepared for use as seed stocks for fermentationcultures. A culture of the transformed recombinant was set up and grownto late log phase (OD₆₀₀=1.0-1.2) in LB broth. The bacteria wereaseptically transferred to centrifuge bottles and centrifuged to pellet.The cells were resuspended in {fraction (1/20)} of the original culturevolume of fresh LB broth. Another {fraction (1/20)} volume of freshLB+20% glycerol was added and the suspension mixed and aliquoted intomultiple 2.0 ml cryotubes. The cultures were allowed to equilibrate inthe glycerol solution for 30 minutes at room temperature, then frozen at−70° C., for long term storage.

[0331] The fermentation was performed as follows. A Bioflo IV fermenterwas sterilized 120 min at 121° C., with dH₂O. The sterile water wasremoved, and fermentation media added as follows:

[0332] 6 liters nitrogen source (100 g yeast extract (BBL) and 200 gtryptone/3 liters (BBL)

[0333] 2 liters 5× fermentation salts

[0334] (48.5 g K₂HPO₄, 12 gm NaH₂PO₄·H₂O, 5 gm NH₄Cl, 2.5 gm NaCI/liter)

[0335] 2 liters 2% glucose

[0336] 20 mls 1 M MgSO₄

[0337] 50 mls 0.05 M CaCl₂

[0338] 2.5-3.5 mls macol P 400 antifoam (PPG Industries Inc., Gurnee,IL)

[0339] 40 mls 10mg/ml kanamycin

[0340] 10 mls trace elements

[0341] (8 g FeSO₄·7H₂O, 2 g MnSO₄·H₂O, 2 g AlCl₃·6H₂O, 0.8 g

[0342] CoCl·6H₂O, 0.4 g ZnSO₄·7H₂O, 0.4 g Na₂MoO₄·2H₂O, 0.2 g

[0343] CuCl₂·2H₂O, 0.2 g NiCl₂, and 0.1 g H₃BO₄/200 mls 5 M HCl)

[0344] All solutions were sterilized by autoclaving, except thekanamycin stock which was filter sterilized.

[0345] Next, 250 μl of glycerol stock was added to the fermenter. Afterseed innoculation, the culture was fermented at 30° C., 125 rpmagitation, and 10 I/min air sparging. The DO₂ control was set to 20%PID, and dissolved oxygen levels were controled by increasing aggitationfrom 125-850 rpm under DO₂ control. DO₂ levels were maintained atgreater than or equal to 20% throughout the entire fermentation. Culturegrowth was continued until endogenous carbon sources were depleted(approximately 12-15 hrs). At this point, a fed batch mode wasinitiated, in which a feed solution of 50% glucose was added at a rateof approximately 4 gm glucose/liter/hr. The pH was adjusted to 7.0 bythe addition of either 25% H₃PO₄ or 4M NaOH solution. Antifoam (a 1:1dilution with filter sterilized 100% ethanol) was added as necessarythroughout the fermentation to prevent foaming. Induction with IPTG (4g) was initiated 2 hrs 40 min after initiation of glucose feed. At 4 hrspost induction, the cells were cooled in the fermentor to 14° C., andstored overnight at 4° C. At the time of induction and at hourlyintervals post induction, 5-10 ml aliquots of cells were harvested.

[0346] Optical density readings were determined by measurement ofabsorbance at 600 nM of 10 μl culture in 990 μl PBS versus a PBScontrol. The density readings of the culture were 47.5, 56, 58, 59, and61.5, at 0-4 hrs post induction, respectively. Cells from each timepointwere serially diluted in PBS (diln 1=1 μ 51 cells/3 ml PBS, diln 2 =15μl diln ⅓ ml PBS, diln 3=6 μl diln ⅔ mls PBS) and 100 μl diln 3 platedon an LB plate and incubated at 37° C. overnight. Cell counts were 154,33 (3), 11 (1), 14 and 25 at 0-4 hrs post induction (bracketed cellcounts represent microcolonies). Morphologically detectable contaminantcolonies were not detected on any plate. LB plates from the uninducedtimepoint were replica plated onto LB+kan, LB+kan+1 mM IPTG and LBplates, in this order. The cultures were grown 6-8 hrs at 37° C. andgrowth on each plate was scored. No colonies were detected on theIPTGKan plate (i.e., no mutations were detected) and 50/50 scoredcolonies were kan resistant (most cells retained plasmid at the time ofinduction).

EXAMPLE 12 QUANTITATIVE ELISA

[0347] In this Example, samples from Example 11 were prepared forquantitative ELISA determination of verotoxin concentration.

[0348] First, soluble extracts were prepared from 10 OD₆₀₀ units ofcells at 0-4 hrs after induction, using cells removed for timepointanalysis, exactly as described in Example 10. An osmotic shockperiplasmic extract from cells at 4 hrs post induction was also preparedas described in Example 10. Briefly, 50 mls of culture were pelleted bycentrifugation 10 min max rpm in a benchtop centrifuge. The pellet wasresuspended in 200 ml 30 mM Tris/20% sucrose pH 8.0 and osmotic shocksolution prepared exactly as described in Example 10.

[0349] An unconcentrated culture supernatant (“unconcentrated culturesoup”) was also tested. After overnight storage at 4° C., cells werepelleted from the entire 10 liter fermentation harvest. This wasperformed by centrifugation at 12,000×g for 10 min, and pooling theclarified culture broth. A 10 ml sample was filtered through a 3 μmfilter; the filtered culture soup was the “unconcentrated culturesupernatant” sample. Finally, a concentrated culture supernatant(“concentrated culture soup”) was also tested. The 10 liter fermentationsupernatant was filtered through a 1.0 μm (Ultipor; Pall) using aperistaltic pump. The supernatant was then filtered through a 0.2 μmfilter (Ultipor; Pall). The resulting 8 1 of cloudy brown supernatantwas concentrated using a preparation-scale TFF 1 ft2 PLGL 10Kregenerated cellulose cartridge (Millipore) connected to a peristalticpump. Material was concentrated down to 0.8 1 and washed with 2 1 of PBS(pH 7.0). A sample of the diafiltration flow through was kept foranalysis. The final concentrated material (650 mls) was aliquoted into50 ml tubes (Falcon) and stored at −70° C. The final concentrated samplewas the concentrated culture supernatant.

[0350] The VT2B concentration in each of these samples was determined byquantitative ELISA measurement as described in Example 10, and theresulting concentrations are shown in the following Table. TABLE 15 VT2BConcentration in pET24VT2B Fermentation Samples Sample VT2BConcentration Total soluble extract: uninduced 50 mg/l Total solubleextract: 1 hr induction 300 mg/l Total soluble extract: 2 hr induction700 mg/l Total soluble extract: 3 hr induction 600 mg/l Total solubleextract: 4 hr induction 600 mg/l Periplasmic extract 100 mg/lUnconcentrated culture soup 750 mg/l Concentrated culture soup 9.4 gm/lCulture soup diafiltration flow-through 3 mg/l

[0351] These results indicated that greater than 50% of the VT2B subunitat harvest was in the culture supernatant. The remainder wascell-associated, of which approximately 20% was periplasmicallylocalized. The culture soup material was concentrated quantitatively bydiafiltration (80% recovery of VT2B in final diafiltered concentratedsample). The losses at the concentration stage were due to diafilterretension, rather than flow through (only 3 mg/l VT2B in diafiltrationflow through). The total yield of VT2B subunit in the concentratedculture supernatant was 6 g from the 10 liter fermentation (startingconcentration was 7.5 g in 10 liters of unconcentrated culturesupernatant). This demonstrated that gram quantities of VT2B subunit canbe generated by fermentation culture of the pET24VT2B BL21(DE3) cellline.

[0352] The final concentrated culture supernatant contained 9.4 mg/mlVT2B, but was heavily discolored with brown material. This coloredmaterial is removed by polyethylenimine (PEI) clarification. To 24 mlsconcentrated culture supernatant 2.7 ml of 2% PEI solution (Mallinkrodt;2% solution in dH₂O, pH 7.5 with HCI) was added, the solution mixed 30min on vibrax and centrifuged 3200×g for 45 min. The concentration ofVT2B in the PEI clarified culture soup was estimated to be 7.3 mg/ml byquantitative ELISA. Thus, PEI clarification can be utilized to removeimpurities from the culture supernatant without precipitating the VT2Bsubunit from solution.

EXAMPLE 13 Purification of Native VT2B Subunit

[0353] In this Example, native VT2B was purified. A PEI clarified,concentrated culture supernatant containing native (nontagged) VT2B wasprepared as in Example 12. A 30 ml aliquot of the supernatant wasfiltered through a Gelman glass fiber Acrodisc 4524 filter, andtransferred to Spectra/Por 3 dialysis tubing (MWCO 3,500). The materialwas dialyzed with mixing one time, overnight, at ambient temperatureagainst 1.5 L of 20 mM sodium phosphate, 0.025% sodium azide, 0.1% Tween20, pH 7.0.

[0354] A 1.5 cm×14 cm column containing Whatman Express-Ion Exchanger C(CM cellulose) was equilibrated with 20 mM sodium phosphate, 0.025%sodium azide, 0.1% Tween 20, pH 7.0. The flow rate was constant at 2ml/minute throughout the following procedure. A 20 ml aliquot of thedialyzed, PEI clarified concentrate containing VT2B was loaded onto thecolumn. The column was washed with 20 mM sodium phosphate, 0.025% sodiumazide, 0.1% Tween 20, pH 7.0. The flow-through and wash were collected(59 ml). The material loaded on the column and the flow-through and washwere assayed by the VT2B Quantitative ELISA as described in Example 10.All of the VT2B loaded onto the column (approximately 150 mg) was in theflow-through and wash. The VT2B was thus partially purified, as manycontaminants bound to the Express-Ion Exchanger C column and wereseparated from the VT2B.

[0355] A 20 ml sample of the Ion Exchanger C flow-through and wash wasloaded onto a 1.5 X 16.5 cm column of Whatman Express-Ion Exchanger Q(QAE cellulose) equilibrated with Buffer A ( 20 mM sodium phosphate, pH7.0. 0.025% sodium azide, 10% glycerol, containing 0.1% each Tween 20,Tween 80 and Hecameg). The flow rate was 2.0 ml/minute throughout thisprocedure. Absorbance at 280 nm was monitored. The column was washed for15 minutes with Buffer A, followed by a linear gradient from 0% Buffer Bto 100% Buffer B over 65 minutes. Buffer B was the same as Buffer A,with the exception that it included 1.5 M sodium chloride.

[0356] The chromatogram was characterized by the following A₂₈₀absorbing material: the flow-through, a small 26 minute peak from 25 to27 minutes, and a large 36 minute peak from 34 to 39 minutes. Thesethree fractions were collected and analyzed in the VT2B QuantitativeELISA, as described in Example 10, and by SDS PAGE (20% polyacrylamide).

[0357] The ELISA results indicated that there was no VT2B activity inthe flow-through or in the small 26 minute peak. However, VT2B activitywas found in the 36 minute peak. The purity of the partially purifiedVT2B was estimated to be 60% on SDS PAGE.

EXAMPLE 14 GENERATION OF VEROTOXIN SUBUNIT IMMUNOGENS

[0358] In this Example, subunit specific antiserum was generatedutilizing purified recombinant verotoxin subunits. As indicated above,the B subunits associate to form a pentamer, and this pentamericconformation was thought to be important for the generation ofneutralizing antibodies. Since the VT2B pentamers may be unstable whenexpressed without the A subunit (See, Acheson et al., Infect. Immun.,63:301 [1995]), crosslinking was utilized to prevent subunitdissociation. The antigens that were utilized for immunization aresummarized in the following Table. In all cases, the antigens utilizedfor immunization are from the recombinant protein preparations describedin Examples ______. TABLE 16 Protein Preparations for ImmunizationVerotoxin Protein Gluteraldehyde Subunit Expression Vector DescriptionCross-Linked VT1A pMalVT1A MBPVT1A No VT2A pMalVT2A MBPVT2A No VT1B(His) pET23HisVT1BL + VT1B (His-tag) No and pET24HisVT1BL + VT2B (His)pET23HisVT2BL + VT2B (His-tag) No and pET24HisVT2BL + VT2BpET24HisVT2BL + VT2B (His-tag) Yes (His + Cross- linked) VT2B (Native)pET24VT2B VT2B (Native) No VT2B pET24VT2B VT2B (Native) Yes (Native +Cross- linked)

[0359] Cross-linking was performed with gluteraldehyde as describedbelow. For VT2B (His) cross-linking, 10 mls of a 1 mg/ml VT2B(His)protein preparation (in imidazole elution buffer [See e.g., Example 8)was dialysed three times for 2 hrs versus a 100-fold excess of PBSutilizing a Pierce 10 K Slide-A-Lyser cassette. To 10 mls of dialysedprotein 1/10 volume (1 ml) of 1% glutaraldehyde (Mallinkrodt) was added,the solution stirred 5 min, and then left overnight at room temperature.The sample was dialysed 2× versus 100 volumes of PBS+ thimerisol forgreater than 6 hrs each dialysis, utilizing a Pierce 10 K Slide-A-Lysercassette. The final dialysed sample was stored at 4° C.

[0360] For VT2B (Native) cross-linking, 10 mls of the concentratedculture supernatant (Example 12) was centrifuged 30 min at 40,000×g topellet insoluble material. The supernatant was dialysed three times for2 hrs versus a 100-fold excess of PBS utilizing a Pierce 10 KSlide-A-Lyser cassette. To 10 mls of dialysed protein {fraction (1/10)}volume (1 ml) of 1% glutaraldehyde (Mallinkrodt) was added, the solutionstirred 5 min, and then left overnight at room temperature. The samplewas dialysed 2× versus 100 volumes of PBS+ thimerisol for greater than 6hrs each dialysis utilizing a Pierce 10 K Slide-A-Lyser cassette. Thefinal dialysed sample was stored at 4° C.

[0361] Samples of untreated and cross-linked material were resolved on a20% SDS-PAGE gel and Coomassie stained, as is shown in FIG. 14.

[0362] In FIG. 14, lanes 1-3 contain VT2B(His) protein preparations,lanes 4-7 contain VT2B(Native) protein preparations, and lane 8 containsbroad range molecular weight markers (Biorad). For gel loading, theVT2B(His) protein preparations were 5 μl protein+5 μl 2× sample buffer,while the VT2B(Native) protein preparations were 0.5 μl protein +4.5 μlPBS+5 μl 2× sample buffer. The lanes are elution (lane 1), dialysedelution (lane 2) and cross-linked elution (lane 3), PEI clarifiedconcentrated culture supernatant (lane 4; Example 12), centrifugedconcentrated culture supernatant (lane 5), dialysed concentrated culturesupernatant (lane 6) and cross-linked concentrated culture supernatant(lane 7).

[0363] The results clearly demonstrated successful cross-linking of theVT2B subunits (both his and native subunits). It is also clear from thegel that the concentrated culture supernatant is highly enriched for theVT2B subunit, as this is the predominant protein band in the sample.

EXAMPLE 15 IMMUNIZATION WITH RECOMBINANT VEROTOXIN SUBUNITS

[0364] In this Example, hens were immunized with purified recombinantsubunit immunogens. Eight groups of white Leghorn laying hens wereinjected subcutaneously with 0.2 - 0.3 mg recombinant verotoxin subunits(pET23hisVT1 BL+, pET23hisVTLBL+ , pMa1VT1 A and pMa1VT2A[BamHI]), mixedwith 5 μg Gerbu, or 75 μg QuilA adjuvants at 2-3 week intervals.

[0365] Eggs were collected from the hens after three or moreimmunizations with verotoxin subunits. Egg yolks were separated fromwhites, pooled and blended with four volumes of 10 mM sodium phosphate,150 mM NaCl, pH 7.4 (PBS). Solid polyethylene glycol 8000 (PEG) was thenadded with mixing, to a final concentration of 3.5% and the mixture wascentrifuged at 10,000×g for 10 minutes. The supernatant was filteredthough cheesecloth and PEG was again added to a final concentration of12% to precipitate the IgY. The solution was centrifuged as above andthe resulting supernatant discarded. The pellet contained the IgY andwas dissolved in PBS to either the original yolk volume (1× PEG IgY)which contained approximately 5 mg/ml IgY or ¼ of the original yolkvolume (4× PEG IgY) which contained approximately 20 mg/ml. Theresuspended IgY was then filtered though a 0.45t membrane and stored at4° C. As a control, eggs from nonimmunized hens or preimmune (PI) henswere harvested and IgY extracted as descibed above.

[0366] To distinguish antibody groups, IgY was named with the antigenfollowed by the initial of the adjuvant used (e.g., “VT1A-G IgY” isantibody produced by hens immunizaed with VT1A using Gerbu adjuvant,whereas “VT1A-Q IgY” was produced by hens immunized with VT1A usingQuil-A).

[0367] ELISAs were used to monitor antibody response during the courseof immunization. IgY's from all immunogen groups were tested againstrVTs. Wells of a microtiter plate were coated with 2.5 μg/ml of rVT's inPBS overnight at 2-80° C. Wells were washed 3 times with PBS containing0.05% Tween-20 (PBS-T), and blocked with PBS containing 5 mg/ml BSA for1 hour at room temperature. 1× PEG IgY from hyperimmune, preimmune eggsand hens immunized with toxoid produced from rVT holotoxin (postivecontrol) was diluted in PBS containing 1 mg/ml BSA, added to the wells,and incubated for lhr at 37° C. Wells were washed as before, andincubated for 1 hr at 37° C. with alkaline phosphatase-conjugated rabbitanti-chicken antibody diluted 1:1000 in PBS-T. Wells were washed againand 1 mg/ml p-nitrophenyl phosphate in 50 mM Na2CO₃, 10 mM MgCl₂ (pH9.5), was added and allowed to incubate at room temperature. Phosphataseactivity was detected by absorbance at 410 mn using a Dynatech MR700microtitier plate reader.

[0368] The validity of each ELISA assay was demonstrated with a positivecontrol using rVT IgY and negative controls using Preimrnune (PI) IgY.The results are given in FIGS. 15-18. VT1 IgY is included in the figuresas a positive control. Preimmune (PI) IgY is included in the figures asa negative control. Titer is defined as binding activity twice as highas PI levels.

[0369]FIG. 15 shows the relatively strong binding of VT1 A-G IgY and VT1A-Q IgY to the homologous toxin rVT1 , with titers of 1:6000 and 1:1200respectively. There was essentially no cross-reactivity of VT2 A-G IgYand VT2 A-Q IgY to VT1 holotoxin.

[0370] As shown in FIG. 16, VT1 A-G IgY and VT1 A-Q IgY cross-reactedstrongly to rVT2; both gave a titer of 1:1200 against rVT2. However thesignal from VT1A-Q IgY was much stronger at the higher concentrations.In contrast, homologous VT2A-Q IgY reactivity to rVT2 gave a much weakerresponse with a titer of 1:250 and VT2A-G IgY did not react over PIlevels.

[0371]FIG. 17 demonstrates the binding of VT1B-G IgY and VT1B-Q IgY torVT1 was similar with titers of 1:500 each. Heterologous VT2B-G IgYbound poorly with a titer of 1:100 while VT2B-Q IgY had a high titer of1:1:2500 to rVT1.

[0372]FIG. 18 shows moderate cross-reactivity of VT1B-G IgY and VTLB-QIgY to VT2, both gave titers of 1:500 and 2500, respectively. Strongreactivity with a titer of 2500 was seen using homologous VT2B-Q IgY toVT2 while VT2B-G IgY showed no significant binding at 1:100.

[0373] In summary, VT1A IgY, VT1 B IgY, and VT2B IgY reacted with bothVT1 and VT2, (i.e., they cross-react). VT2A IgY reacted only with VT2holotoxin. Overall, antibodies from animals immunized with QuilAperformed better than those from animals immunized with Gerbu. Inaddition, QuilA is a more economical adjuvant, costing approximately 5times less per immunization than Gerbu.

EXAMPLE 16 TOXIN NEUTRALIZATION: CHALLENGE IN MICE

[0374] In this Example, protection experiments were performed in mice,in a manner similar to that of Example 5. Two aspects of this experimentare included, the Toxin Challenge Model and the Viable OrganismInfection Model.

[0375] A. Toxin Challenge Model

[0376] rVT1 or rVT2 was premixed with IgY and injected into mice todetermine whether toxin could be neutralized. Tables 17-23 show theresults of rTV1 neutralization studies. VT1 A-G IgY, VT1A-Q IgY, and VT1B-Q IgY all successfully neutralized rTV1 . VT2A-G IgY, VT2A-Q IgY, VT1B-G IgY, and VT2B-Q IgY did not protect the mice. Tables 24-27 summarizethe rVT2 neutralization studies. Only VT2B-Q IgY was capable ofpreventing lethality by rVT2. The other antibodies tested, VT1A-G IgY,VT1 A-Q IgY, VT2A-G IgY, and VT2B-Q IgY were unable to neutralize thetoxin. Therefore neutralizing antibodies to both toxins were generated,though no cross-neutralization was found. In this table, “N.S.”indicates that there were no statistically significant differences(Chi-square analysis). TABLE 17 Neutralization of rVT1 Using VT1A-G IgYand VT1-Q IgY IgY Results of Individual Trials Sum of All TrialsAntibody Tested #survivors/#total #Survivors/#Total p Preimmune 0/6 3/13 Antibody 3/7 VT1A-G IgY 7/7 14/14 <.001 7/7 VT1A-Q IgY 6/6 11/13<.01  5/7

[0377] TABLE 18 Neutralization of rVT1 Using VT2A-G IgY Antibody Tested#Survivors/#Total p Preimmune IgY 0/6 VT2A-G IgY 1/6 N.S.

[0378] TABLE 19 Neutralization of rVT1 Using VT2A-Q IgY Antibody Tested#Survivors/#Total p Preimmune IgY 3/7 VT2A-Q IgY 2/7 N.S.

[0379] TABLE 20 Results for VT1B-Q IgY Antibody Tested #Survivors/#Totalp Preimmune IgY 3/7 T1B-G IgY 7/7 N.S.

[0380] While the results in Table 20 indicate no statisticallysignificant difference, it is assumed from the successful neutralizationof anti-VT1 B-Q, that anti-VTB-G should be effective. TABLE 21Neutralization of rVT1 Using VT1B-Q IgY Results of Antibody IndividualTrials Sum of All Trials Tested #survivors/#total #Survivors/#Total pPreimmune 3/9  3/14 IgY VT1B-Q IgY 9/9 14/14 <0.001

[0381] TABLE 22 Neutralization of rVT1 Using VT2B-G IgY Antibody Tested#Survivors/#Total p Preimmune IgY 3/7 VT2B-G IgY 2/7 N.S.

[0382] TABLE 23 Neutralization of rVT1 Using VT2B-Q IgY Sum of AllResults of Individual Trials Trials #Survivors/ Antibody Tested#Survivors/#Total #Total p Preimmune IgY 2/7 3/14 1/7 VT2B-Q IgY 3/73/14 N.S. 0/7

[0383] TABLE 24 Neutralization of rVT2 Using VT1A-G IgY Results ofIndividual Trials Antibody Tested #Survivors/#Total p Preimmune IgY 4/7VT1A-G IgY 3/6 N.S.

[0384] TABLE 25 Neutralization of rVT2 Using VT1A-Q IgY Antibody Tested#Survivors/#Total p Preimmune IgY 1/7 VT1A-Q IgY 2/7 N.S.

[0385] TABLE 26 Neutralization of rVT2 Using VT2A-G IgY Sum of allResults of Individual Trials Trials #Survivors/ Antibody Tested#Survivors/#Total #Total p Preimmune IgY 4/7 5/14 1/7 VT2A-G IgY 2/64/13 N.S. 2/7

[0386] TABLE 27 Neutralization of rVT2 Using VT1B-Q IgY and VT2B-Q IgYSum of All Results of Individual Trials Trials #Survivors/ AntibodyTested #Survivors/#Total #Total p Preimmune IgY 1/7 1/7 VT1B-Q IgY 0/70/7 N.S. VT2B-Q IgY 7/7 7/7 <0.01

[0387] B. Viable Organism Infection Model

[0388] The toxin neutralizing ability of VT IgY was further demonstratedin an infection study. This experiment was performed as described inExample 5. In this Example, mice were infected with either E. coliO91:H21 (strain B2F1) Because the infecting organism produces a variantof VT2 (ie., VT2c), only antibodies demonstrating neutralizing abilityto VT2 toxin in vitro were tested. For the results shown in Table 28,IgY was administered intraperitoneally at 4 and 10 hours post-infectionand once a day thereafter for the next three days. For the results shownin Table 29, IgY was administered intraperitoneally at 4 hourspost-infection and once a day thereafter for the next three days.

[0389] The results shown in Tables 28 and 29 demonstrate that the VT2B-QIgY protected mice from lethality when administered 4 hrs followinginfection (the longest treatment window tested) and that preimmune IgYwas unable to protect the animals.

[0390] These results indicated that IgY capable of neutralizing VT2 wasgenerated and therefore, the VT2B-Q IgY provides therapeutic benefit forthe treatment of VTEC infections. TABLE 28 Protection of Mice from E.coli 091:H21 with VT2B-Q IgY IgY Treatment Survivors/Total p PreimmuneAntibody 2/10 VT2B-Q IgY 8/10 <0.05

[0391] TABLE 29 Protection of Mice from E. coli 091:H21 with VT2B-Q IgYIgY Treatment Survivors/Total p Preimmune Antibody 0/10 VT2B-Q IgY 9/10<0.001

EXAMPLE 17 ANTI-VEROTOXIN PRODUCTION IN RABBITS

[0392] In this Example, rabbits were used to produce neutralizingantibodies against VT1 and VT2.

[0393] Purified VT1B (pET24hisVT1 BL+), VT2B (pET24hisVT2BL+), VT2A(pMa1VT2A[BamHI]), VT2B (His+Cross-linked), VT2B(Native+Cross-linked),and VT2B (Native, concentrated) were used as immunogens in thisexperiment. As with earler examples, to distinguish antibody groups, IgYwas named with the antigen follwed by the initial adjuvant used (e.g.,“VT1A-G Ig” is antibody produced by rabbits immunized with VT1A usingGerbu adjuvant, and “VT1A-A Ig” is antibody produced by rabbitsimmunized with VT1A using alum adjuvant). These antigens were preparedas listed in Table 16, with the exception being rVT1 A, which was notused.

[0394] A group of New Zealand rabbits were immunized IM a one site with500 μg VT1B and VT2B, mixed with 10 μg Gerbu, or an equal volume ofalum. One month later, the rabbits were boosted, by reinjecting in thesame manner. Two weeks after the boots, the rabbits were bled. The bloodwas sera stored at 37° C. for 1 hour, and centrifuged. The sera werecollected, and stored at −20° C. until testing, as described below.

[0395] A second group of rabbits was initially immunized ID, in at least20 sites with 300 μg VT2B (His+Cross-linked), VT2B(Native+Cross-linked), and VT2B (Native), with 100 μg QuilA, or an equalamount of alum. Another group of rabbits was immunized with 90 μg ofVT2A and the same volumes of adjuvant as described above. One monthafter primary immunization, the rabbits were boosted SC, and were bledtwo weeks after the boost. These sera were collected and stored asdescribed above.

EXAMPLE 18 ELISA TESTING OF RABBIT SERA

[0396] In this Example, the sera collected from the rabbits described inExample 17 were tested in an ELISA. In this experiment, the ELISAmethods used in previous examples (See, Example 10) were used, with theexception being that the primary antibody was rabbit sera and thesecondary antibody was goat anti-rabbit (1:1500 dilution).

[0397] The focus of this Example, were ELISAs that tested the bindingability of Ig to native toxin. Only VT1B-A Ig, and VT1B-G Ig reacted torVT1 at dilutions of 1:2500. Neither VT2B-A Ig, VT2B-G Ig, nor preimmuneIg reacted with rVT1 at a dilution greater than 1:100. For rVT2, only VT1 B-A Ig showed a specific antibody response, with the Ig reacting at adilution of 1:2500.

EXAMPLE 19 TOXIN CHALLENGE MODEL

[0398] In this Example, the ability of rabbit Ig produced as describedin Example 17 was tested for its ability to protect mice from theeffects of verotoxin.

[0399] Sera was premixed with a lethal dose of toxin (lethal dose wasdetermined as described in Ex. iD). This preparation was then injectedIP into mice. The mice were observed for seven days post injection asdescribed in Ex. SA. The results are summarized in Tables 30-33.

[0400] The results shown in Table 30 indicate that both VT1 B-A Ig andVT1 B-G Ig completely protected the mice against rVT1 when tested in thesystem shown in the table. However, VT2B-A Ig did not protect the micefrom lethality. The results in Table 31 show that none of the antibodiestested as shown in this table protected against rVT2. The results inTable 32 show that VT2B(His+Cross-linked) Ig, VT2B(Native+Cross-linked)Ig, completely neutralized rVT2, regardless of the adjuvant used. Inaddition, VT2A Ig provided some protection. The results in Table 33 showthat neither VT2B(native)-A Ig, nor VT2B(Native)-Q Ig providedstatistically significant protection against rVT2. TABLE 30 3 LD₅₀ rVT1Premixed with Serum (Final Dilution 1:100) Antibody Tested#Survivors/#Total Preimmune Ig  1/14 VT1B-A Ig 14/14 VT1B-G Ig 14/14VT2B-A Ig 2/7

[0401] TABLE 31 7 LD₅₀s of rVT2 Premixed with Serum (Final Dilution1:100) Antibody Tested #Survivors/#Total Preimmune Ig 1/14 VT1B-A Ig0/14 VT1B-G Ig 2/14 VT2B-A Ig 1/14 VT2B-G Ig 3/14

[0402] TABLE 32 3 LD₅₀s of rVT1 Premixed with Serum (Final Dilution1:100) Antibody Tested #Survivors/#Total Preimmune Ig 3/7 VT2B(His +Cross-linked)-A Ig 2/7 VT2B(His + Cross-linked)-Q Ig 2/7 VT2B(Native +Cross-linked)-A Ig 0/7 VT2B(Native + Cross-linked)-Q Ig 3/7 VT2A-A Ig4/7 VT2A-Q Ig 4/7

[0403] TABLE 33 LD₅₀s of rVT2 Premixed with Serum (Final Dilution 1:100)Antibody Tested # Survivors/# Total Preimmune Ig 0/7 VT2B(His +Cross-linked)-A Ig 7/7 VT2B(His + Cross-linked)-Q Ig 7/7 VT2B(Native +Cross-linked)-A Ig 7/7 VT2B(Native + Cross-linked)-Q Ig 7/7 VT2A-A Ig5/7 VT2A-Q Ig 3/7

[0404] TABLE 34 LD₅₀ rVT2 Premixed with Serum (Final Dilution 1:100)Antibody Tested # Survivors/# Total Preimmune Ig 1/6 VT2B(Native)-A Ig5/6 VT2B(Native)-Q Ig 4/6

EXAMPLE 20 rVT2B Column

[0405] In this Example, rVT2B was covalently attached to an aldehydeactivated agarose matrix. One ml VT2B was mixed with 50 μl Actigel resin(Sterogene), and 200 Ill of coupling solution (Sterogene). The mixturewas incuabed overnight with agitation and poured into a column. Thecolumn was equilibrated with PBS containing 0.005% thimerosol.Fifty-five ml of a VT2B-Q IgY PEG preparation or a VT2B-G IgY PEGpreparation prepared as described above were applied to the column. Theflow-through was collected and reloaded onto the column. The column wasthen washed with PBS containing 0.005% thimerosol until baseline wasre-established. Bound antibody was eluted from the column in Actisepelution buffer (Sterogene), and the elution peak was collected. Thecolumn was re-equilibrated.

[0406] Affinity-purified specific antibodies were dialyzed against threechanges of a 130-fold volume of PBC 4° C. The percentage of specificVT2B IgY present in each preparation was determined using UV absorbanceand expressed as a percentage of total protein in the preparation.Purification of VT2B-Q IgY yielded 0.6% specific antibody andpurification of VT2B-G IgY was approximately 0.2% specific antibody.

[0407] From the above, it is clear that the present invention providescompositions and methods for the preparation of effective multivalentvaccines against Escherichia coli verotoxins. It is also contemplatedthat the recombinant verotoxin proteins be used for the production ofantitoxins. All publications and patents mentioned in the abovespecification are herein incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention.

1 49 945 base pairs nucleic acid double linear DNA (genomic) CDS 1..9451 ATG AAA ATA ATT ATT TTT AGA GTG CTA ACT TTT TTC TTT GTT ATC TTT 48 MetLys Ile Ile Ile Phe Arg Val Leu Thr Phe Phe Phe Val Ile Phe 1 5 10 15TCA GTT AAT GTG GTG GCG AAG GAA TTT ACC TTA GAC TTC TCG ACT GCA 96 SerVal Asn Val Val Ala Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala 20 25 30 AAGACG TAT GTA GAT TCG CTG AAT GTC ATT CGC TCT GCA ATA GGT ACT 144 Lys ThrTyr Val Asp Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr 35 40 45 CCA TTACAG ACT ATT TCA TCA GGA GGT ACG TCT TTA CTG ATG ATT GAT 192 Pro Leu GlnThr Ile Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp 50 55 60 AGT GGC TCAGGG GAT AAT TTG TTT GCA GTT GAT GTC AGA GGG ATA GAT 240 Ser Gly Ser GlyAsp Asn Leu Phe Ala Val Asp Val Arg Gly Ile Asp 65 70 75 80 GCA GAG GAAGGG CGG TTT AAT AAT CTA CGG CTT ATT GTT GAA CGA AAT 288 Ala Glu Glu GlyArg Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn 85 90 95 AAT TTA TAT GTGACA GGA TTT GTT AAC AGG ACA AAT AAT GTT TTT TAT 336 Asn Leu Tyr Val ThrGly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr 100 105 110 CGC TTT GCT GATTTT TCA CAT GTT ACC TTT CCA GGT ACA ACA GCG GTT 384 Arg Phe Ala Asp PheSer His Val Thr Phe Pro Gly Thr Thr Ala Val 115 120 125 ACA TTG TCT GGTGAC AGT AGC TAT ACC ACG TTA CAG CGT GTT GCA GGG 432 Thr Leu Ser Gly AspSer Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly 130 135 140 ATC AGT CGT ACGGGG ATG CAG ATA AAT CGC CAT TCG TTG ACT ACT TCT 480 Ile Ser Arg Thr GlyMet Gln Ile Asn Arg His Ser Leu Thr Thr Ser 145 150 155 160 TAT CTG GATTTA ATG TCG CAT AGT GGA ACC TCA CTG ACG CAG TCT GTG 528 Tyr Leu Asp LeuMet Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val 165 170 175 GCA AGA GCGATG TTA CGG TTT GTT ACT GTG ACA GCT GAA GCT TTA CGT 576 Ala Arg Ala MetLeu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg 180 185 190 TTT CGG CAAATA CAG AGG GGA TTT CGT ACA ACA CTG GAT GAT CTC AGT 624 Phe Arg Gln IleGln Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser 195 200 205 GGG CGT TCTTAT GTA ATG ACT GCT GAA GAT GTT GAT CTT ACA TTG AAC 672 Gly Arg Ser TyrVal Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn 210 215 220 TGG GGA AGGTTG AGT AGC GTC CTG CCT GAC TAT CAT GGA CAA GAC TCT 720 Trp Gly Arg LeuSer Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser 225 230 235 240 GTT CGTGTA GGA AGA ATT TCT TTT GGA AGC ATT AAT GCA ATT CTG GGA 768 Val Arg ValGly Arg Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly 245 250 255 AGC GTGGCA TTA ATA CTG AAT TGT CAT CAT CAT GCA TCG CGA GTT GCC 816 Ser Val AlaLeu Ile Leu Asn Cys His His His Ala Ser Arg Val Ala 260 265 270 AGA ATGGCA TCT GAT GAG TTT CCT TCT ATG TGT CCG GCA GAT GGA AGA 864 Arg Met AlaSer Asp Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg 275 280 285 GTC CGTGGG ATT ACG CAC AAT AAA ATA TTG TGG GAT TCA TCC ACT CTG 912 Val Arg GlyIle Thr His Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu 290 295 300 GGG GCAATT CTG ATG CGC AGA ACT ATT AGC AGT 945 Gly Ala Ile Leu Met Arg Arg ThrIle Ser Ser 305 310 315 315 amino acids amino acid linear protein 2 MetLys Ile Ile Ile Phe Arg Val Leu Thr Phe Phe Phe Val Ile Phe 1 5 10 15Ser Val Asn Val Val Ala Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala 20 25 30Lys Thr Tyr Val Asp Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr 35 40 45Pro Leu Gln Thr Ile Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp 50 55 60Ser Gly Ser Gly Asp Asn Leu Phe Ala Val Asp Val Arg Gly Ile Asp 65 70 7580 Ala Glu Glu Gly Arg Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn 85 9095 Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr 100105 110 Arg Phe Ala Asp Phe Ser His Val Thr Phe Pro Gly Thr Thr Ala Val115 120 125 Thr Leu Ser Gly Asp Ser Ser Tyr Thr Thr Leu Gln Arg Val AlaGly 130 135 140 Ile Ser Arg Thr Gly Met Gln Ile Asn Arg His Ser Leu ThrThr Ser 145 150 155 160 Tyr Leu Asp Leu Met Ser His Ser Gly Thr Ser LeuThr Gln Ser Val 165 170 175 Ala Arg Ala Met Leu Arg Phe Val Thr Val ThrAla Glu Ala Leu Arg 180 185 190 Phe Arg Gln Ile Gln Arg Gly Phe Arg ThrThr Leu Asp Asp Leu Ser 195 200 205 Gly Arg Ser Tyr Val Met Thr Ala GluAsp Val Asp Leu Thr Leu Asn 210 215 220 Trp Gly Arg Leu Ser Ser Val LeuPro Asp Tyr His Gly Gln Asp Ser 225 230 235 240 Val Arg Val Gly Arg IleSer Phe Gly Ser Ile Asn Ala Ile Leu Gly 245 250 255 Ser Val Ala Leu IleLeu Asn Cys His His His Ala Ser Arg Val Ala 260 265 270 Arg Met Ala SerAsp Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg 275 280 285 Val Arg GlyIle Thr His Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu 290 295 300 Gly AlaIle Leu Met Arg Arg Thr Ile Ser Ser 305 310 315 267 base pairs nucleicacid double linear DNA (genomic) CDS 1..267 3 ATG AAA AAA ACA TTA TTAATA GCT GCA TCG CTT TCA TTT TTT TCA GCA 48 Met Lys Lys Thr Leu Leu IleAla Ala Ser Leu Ser Phe Phe Ser Ala 1 5 10 15 AGT GCG CTG GCG ACG CCTGAT TGT GTA ACT GGA AAG GTG GAG TAT ACA 96 Ser Ala Leu Ala Thr Pro AspCys Val Thr Gly Lys Val Glu Tyr Thr 20 25 30 AAA TAT AAT GAT GAC GAT ACCTTT ACA GTT AAA GTG GGT GAT AAA GAA 144 Lys Tyr Asn Asp Asp Asp Thr PheThr Val Lys Val Gly Asp Lys Glu 35 40 45 TTA TTT ACC AAC AGA TGG AAT CTTCAG TCT CTT CTT CTC AGT GCG CAA 192 Leu Phe Thr Asn Arg Trp Asn Leu GlnSer Leu Leu Leu Ser Ala Gln 50 55 60 ATT ACG GGG ATG ACT GTA ACC ATT AAAACT AAT GCC TGT CAT AAT GGA 240 Ile Thr Gly Met Thr Val Thr Ile Lys ThrAsn Ala Cys His Asn Gly 65 70 75 80 GGG GGA TTC AGC GAA GTT ATT TTT CGT267 Gly Gly Phe Ser Glu Val Ile Phe Arg 85 89 amino acids amino acidlinear protein 4 Met Lys Lys Thr Leu Leu Ile Ala Ala Ser Leu Ser Phe PheSer Ala 1 5 10 15 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly Lys ValGlu Tyr Thr 20 25 30 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val GlyAsp Lys Glu 35 40 45 Leu Phe Thr Asn Arg Trp Asn Leu Gln Ser Leu Leu LeuSer Ala Gln 50 55 60 Ile Thr Gly Met Thr Val Thr Ile Lys Thr Asn Ala CysHis Asn Gly 65 70 75 80 Gly Gly Phe Ser Glu Val Ile Phe Arg 85 954 basepairs nucleic acid double linear DNA (genomic) CDS 1..954 5 ATG AAG TGTATA TTA TTT AAA TGG GTA CTG TGC CTG TTA CTG GGT TTT 48 Met Lys Cys IleLeu Phe Lys Trp Val Leu Cys Leu Leu Leu Gly Phe 1 5 10 15 TCT TCG GTATCC TAT TCC CGG GAG TTT ACG ATA GAC TTT TCG ACC CAA 96 Ser Ser Val SerTyr Ser Arg Glu Phe Thr Ile Asp Phe Ser Thr Gln 20 25 30 CAA AGT TAT GTCTCT TCG TTA AAT AGT ATA CGG ACA GAG ATA TCG ACC 144 Gln Ser Tyr Val SerSer Leu Asn Ser Ile Arg Thr Glu Ile Ser Thr 35 40 45 CCT CTT GAA CAT ATATCT CAG GGG ACC ACA TCG GTG TCT GTT ATT AAC 192 Pro Leu Glu His Ile SerGln Gly Thr Thr Ser Val Ser Val Ile Asn 50 55 60 CAC ACC CAC GGC AGT TATTTT GCT GTG GAT ATA CGA GGG CTT GAT GTC 240 His Thr His Gly Ser Tyr PheAla Val Asp Ile Arg Gly Leu Asp Val 65 70 75 80 TAT CAG GCG CGT TTT GACCAT CTT CGT CTG ATT ATT GAG CAA AAT AAT 288 Tyr Gln Ala Arg Phe Asp HisLeu Arg Leu Ile Ile Glu Gln Asn Asn 85 90 95 TTA TAT GTG GCA GGG TTC GTTAAT ACG GCA ACA AAT ACT TTC TAC CGT 336 Leu Tyr Val Ala Gly Phe Val AsnThr Ala Thr Asn Thr Phe Tyr Arg 100 105 110 TTT TCA GAT TTT ACA CAT ATATCA GTG CCC GGT GTG ACA ACG GTT TCC 384 Phe Ser Asp Phe Thr His Ile SerVal Pro Gly Val Thr Thr Val Ser 115 120 125 ATG ACA ACG GAC AGC AGT TATACC ACT CTG CAA CGT GTC GCA GCG CTG 432 Met Thr Thr Asp Ser Ser Tyr ThrThr Leu Gln Arg Val Ala Ala Leu 130 135 140 GAA CGT TCC GGA ATG CAA ATCAGT CGT CAC TCA CTG GTT TCA TCA TAT 480 Glu Arg Ser Gly Met Gln Ile SerArg His Ser Leu Val Ser Ser Tyr 145 150 155 160 CTG GCG TTA ATG GAG TTCAGT GGT AAT ACA ATG ACC AGA GAT GCA TCC 528 Leu Ala Leu Met Glu Phe SerGly Asn Thr Met Thr Arg Asp Ala Ser 165 170 175 AGA GCA GTT CTG CGT TTTGTC ACT GTC ACA GCA GAA GCC TTA CGC TTC 576 Arg Ala Val Leu Arg Phe ValThr Val Thr Ala Glu Ala Leu Arg Phe 180 185 190 AGG CAG ATA CAG AGA GAATTT CGT CAG GCA CTG TCT GAA ACT GCT CCT 624 Arg Gln Ile Gln Arg Glu PheArg Gln Ala Leu Ser Glu Thr Ala Pro 195 200 205 GTG TAT ACG ATG ACG CCGGGA GAC GTG GAC CTC ACT CTG AAC TGG GGG 672 Val Tyr Thr Met Thr Pro GlyAsp Val Asp Leu Thr Leu Asn Trp Gly 210 215 220 CGA ATC AGC AAT GTG CTTCCG GAG TAT CGG GGA GAG GAT GGT GTC AGA 720 Arg Ile Ser Asn Val Leu ProGlu Tyr Arg Gly Glu Asp Gly Val Arg 225 230 235 240 GTG GGG AGA ATA TCCTTT AAT AAT ATA TCA GCG ATA CTG GGG ACT GTG 768 Val Gly Arg Ile Ser PheAsn Asn Ile Ser Ala Ile Leu Gly Thr Val 245 250 255 GCC GTT ATA CTG AATTGC CAT CAT CAG GGG GCG CGT TCT GTT CGC GCC 816 Ala Val Ile Leu Asn CysHis His Gln Gly Ala Arg Ser Val Arg Ala 260 265 270 GTG AAT GAA GAG AGTCAA CCA GAA TGT CAG ATA ACT GGC GAC AGG CCT 864 Val Asn Glu Glu Ser GlnPro Glu Cys Gln Ile Thr Gly Asp Arg Pro 275 280 285 GTT ATA AAA ATA AACAAT ACA TTA TGG GAA AGT AAT ACA GCT GCA GCG 912 Val Ile Lys Ile Asn AsnThr Leu Trp Glu Ser Asn Thr Ala Ala Ala 290 295 300 TTT CTG AAC AGA AAGTCA CAG TTT TTA TAT ACA ACG GGT AAA 954 Phe Leu Asn Arg Lys Ser Gln PheLeu Tyr Thr Thr Gly Lys 305 310 315 318 amino acids amino acid linearprotein 6 Met Lys Cys Ile Leu Phe Lys Trp Val Leu Cys Leu Leu Leu GlyPhe 1 5 10 15 Ser Ser Val Ser Tyr Ser Arg Glu Phe Thr Ile Asp Phe SerThr Gln 20 25 30 Gln Ser Tyr Val Ser Ser Leu Asn Ser Ile Arg Thr Glu IleSer Thr 35 40 45 Pro Leu Glu His Ile Ser Gln Gly Thr Thr Ser Val Ser ValIle Asn 50 55 60 His Thr His Gly Ser Tyr Phe Ala Val Asp Ile Arg Gly LeuAsp Val 65 70 75 80 Tyr Gln Ala Arg Phe Asp His Leu Arg Leu Ile Ile GluGln Asn Asn 85 90 95 Leu Tyr Val Ala Gly Phe Val Asn Thr Ala Thr Asn ThrPhe Tyr Arg 100 105 110 Phe Ser Asp Phe Thr His Ile Ser Val Pro Gly ValThr Thr Val Ser 115 120 125 Met Thr Thr Asp Ser Ser Tyr Thr Thr Leu GlnArg Val Ala Ala Leu 130 135 140 Glu Arg Ser Gly Met Gln Ile Ser Arg HisSer Leu Val Ser Ser Tyr 145 150 155 160 Leu Ala Leu Met Glu Phe Ser GlyAsn Thr Met Thr Arg Asp Ala Ser 165 170 175 Arg Ala Val Leu Arg Phe ValThr Val Thr Ala Glu Ala Leu Arg Phe 180 185 190 Arg Gln Ile Gln Arg GluPhe Arg Gln Ala Leu Ser Glu Thr Ala Pro 195 200 205 Val Tyr Thr Met ThrPro Gly Asp Val Asp Leu Thr Leu Asn Trp Gly 210 215 220 Arg Ile Ser AsnVal Leu Pro Glu Tyr Arg Gly Glu Asp Gly Val Arg 225 230 235 240 Val GlyArg Ile Ser Phe Asn Asn Ile Ser Ala Ile Leu Gly Thr Val 245 250 255 AlaVal Ile Leu Asn Cys His His Gln Gly Ala Arg Ser Val Arg Ala 260 265 270Val Asn Glu Glu Ser Gln Pro Glu Cys Gln Ile Thr Gly Asp Arg Pro 275 280285 Val Ile Lys Ile Asn Asn Thr Leu Trp Glu Ser Asn Thr Ala Ala Ala 290295 300 Phe Leu Asn Arg Lys Ser Gln Phe Leu Tyr Thr Thr Gly Lys 305 310315 267 base pairs nucleic acid double linear DNA (genomic) CDS 1..267 7ATG AAG AAG ATG TTT ATG GCG GTT TTA TTT GCA TTA GCT TCT GTT AAT 48 MetLys Lys Met Phe Met Ala Val Leu Phe Ala Leu Ala Ser Val Asn 1 5 10 15GCA ATG GCG GCG GAT TGT GCT AAA GGT AAA ATT GAG TTT TCC AAG TAT 96 AlaMet Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr 20 25 30 AATGAG GAT GAC ACA TTT ACA GTG AAG GTT GAC GGG AAA GAA TAC TGG 144 Asn GluAsp Asp Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp 35 40 45 ACC AGTCGC TGG AAT CTG CAA CCG TTA CTG CAA AGT GCT CAG TTG ACA 192 Thr Ser ArgTrp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr 50 55 60 GGA ATG ACTGTC ACA ATC AAA TCC AGT ACC TGT GAA TCA GGC TCC GGA 240 Gly Met Thr ValThr Ile Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly 65 70 75 80 TTT GCT GAAGTG CAG TTT AAT AAT GAC 267 Phe Ala Glu Val Gln Phe Asn Asn Asp 85 89amino acids amino acid linear protein 8 Met Lys Lys Met Phe Met Ala ValLeu Phe Ala Leu Ala Ser Val Asn 1 5 10 15 Ala Met Ala Ala Asp Cys AlaLys Gly Lys Ile Glu Phe Ser Lys Tyr 20 25 30 Asn Glu Asp Asp Thr Phe ThrVal Lys Val Asp Gly Lys Glu Tyr Trp 35 40 45 Thr Ser Arg Trp Asn Leu GlnPro Leu Leu Gln Ser Ala Gln Leu Thr 50 55 60 Gly Met Thr Val Thr Ile LysSer Ser Thr Cys Glu Ser Gly Ser Gly 65 70 75 80 Phe Ala Glu Val Gln PheAsn Asn Asp 85 1241 base pairs nucleic acid double linear DNA (genomic)9 ATGAAAATAA TTATTTTTAG AGTGCTAACT TTTTTCTTTG TTATCTTTTC AGTTAATGTG 60GTGGCGAAGG AATTTACCTT AGACTTCTCG ACTGCAAAGA CGTATGTAGA TTCGCTGAAT 120GTCATTCGCT CTGCAATAGG TACTCCATTA CAGACTATTT CATCAGGAGG TACGTCTTTA 180CTGATGATTG ATAGTGGCTC AGGGGATAAT TTGTTTGCAG TTGATGTCAG AGGGATAGAT 240GCAGAGGAAG GGCGGTTTAA TAATCTACGG CTTATTGTTG AACGAAATAA TTTATATGTG 300ACAGGATTTG TTAACAGGAC AAATAATGTT TTTTATCGCT TTGCTGATTT TTCACATGTT 360ACCTTTCCAG GTACAACAGC GGTTACATTG TCTGGTGACA GTAGCTATAC CACGTTACAG 420CGTGTTGCAG GGATCAGTCG TACGGGGATG CAGATAAATC GCCATTCGTT GACTACTTCT 480TATCTGGATT TAATGTCGCA TAGTGGAACC TCACTGACGC AGTCTGTGGC AAGAGCGATG 540TTACGGTTTG TTACTGTGAC AGCTGAAGCT TTACGTTTTC GGCAAATACA GAGGGGATTT 600CGTACAACAC TGGATGATCT CAGTGGGCGT TCTTATGTAA TGACTGCTGA AGATGTTGAT 660CTTACATTGA ACTGGGGAAG GTTGAGTAGC GTCCTGCCTG ACTATCATGG ACAAGACTCT 720GTTCGTGTAG GAAGAATTTC TTTTGGAAGC ATTAATGCAA TTCTGGGAAG CGTGGCATTA 780ATACTGAATT GTCATCATCA TGCATCGCGA GTTGCCAGAA TGGCATCTGA TGAGTTTCCA 840TCTATGTGTC CGGCAGATGG AAGAGTCCGT GGGATTACGC ACAATAAAAT ATTGTGGGAT 900TCATCCACTC TGGGGGCAAT TCTGATGCGC AGAACTATTA GCAGTTGAAC AGGGGGTAAA 960TAAAGGAGTT AAGCATGAAA AAAACATTAT TAATAGCTGC ATCGCTTTCA TTTTTTTCAG 1020CAAGTGCGCT GGCGACGCCT GATTGTGTAA CTGGAAAGGT GGAGTATACA AAATATAATG 1080ATGACGATAC CTTTACAGTT AAAGTGGGTG ATAAAGAATT ATTTACCAAC AGATGGAATC 1140TTCAGTCTCT TCTTCTCAGT GCGCAAATTA CGGGGATGAC TGTAACCATT AAAACTAATG 1200CCTGTCATAA TGGAGGGGGA TTCAGCGAAG TTATTTTTCG T 1241 1235 base pairsnucleic acid double linear DNA (genomic) 10 ATGAAGTGTA TATTATTTAAATGGGTACTG TGCCTGTTAC TGGGTTTTTC TTCGGTATCC 60 TATTCCCGGG AGTTTACGATAGACTTTTCG ACCCAACAAA GTTATGTCTC TTCGTTAAAT 120 AGTATACGGA CAGAGATATCGACCCCTCTT GAACATATAT CTCAGGGGAC CACATCGGTG 180 TCTGTTATTA ACCACACCCACGGCAGTTAT TTTGCTGTGG ATATACGAGG GCTTGATGTC 240 TATCAGGCGC GTTTTGACCATCTTCGTCTG ATTATTGAGC AAAATAATTT ATATGTGGCA 300 GGGTTCGTTA ATACGGCAACAAATACTTTC TACCGTTTTT CAGATTTTAC ACATATATCA 360 GTGCCCGGTG TGACAACGGTTTCCATGACA ACGGACAGCA GTTATACCAC TCTGCAACGT 420 GTCGCAGCGC TGGAACGTTCCGGAATGCAA ATCAGTCGTC ACTCACTGGT TTCATCATAT 480 CTGGCGTTAA TGGAGTTCAGTGGTAATACA ATGACCAGAG ATGCATCCAG AGCAGTTCTG 540 CGTTTTGTCA CTGTCACAGCAGAAGCCTTA CGCTTCAGGC AGATACAGAG AGAATTTCGT 600 CAGGCACTGT CTGAAACTGCTCCTGTGTAT ACGATGACGC CGGGAGACGT GGACCTCACT 660 CTGAACTGGG GGCGAATCAGCAATGTGCTT CCGGAGTATC GGGGAGAGGA TGGTGTCAGA 720 GTGGGGAGAA TATCCTTTAATAATATATCA GCGATACTGG GGACTGTGGC CGTTATACTG 780 AATTGCCATC ATCAGGGGGCGCGTTCTGTT CGCGCCGTGA ATGAAGAGAG TCAACCAGAA 840 TGTCAGATAA CTGGCGACAGGCCTGTTATA AAAATAAACA ATACATTATG GGAAAGTAAT 900 ACAGCTGCAG CGTTTCTGAACAGAAAGTCA CAGTTTTTAT ATACAACGGG TAAATAAAGG 960 AGTTAAGCAT GAAGAAGATGTTTATGGCGG TTTTATTTGC ATTAGCTTCT GTTAATGCAA 1020 TGGCGGCGGA TTGTGCTAAAGGTAAAATTG AGTTTTCCAA GTATAATGAG GATGACACAT 1080 TTACAGTGAA GGTTGACGGGAAAGAATACT GGACCAGTCG CTGGAATCTG CAACCGTTAC 1140 TGCAAAGTGC TCAGTTGACAGGAATGACTG TCACAATCAA ATCCAGTACC TGTGAATCAG 1200 GCTCCGGATT TGCTGAAGTGCAGTTTAATA ATGAC 1235 8 amino acids amino acid unknown linear peptide 11Leu Glu His His His His His His 1 5 29 base pairs nucleic acid singlelinear DNA (genomic) 12 GCCATATGAA AATAATTATT TTTAGAGTG 29 29 base pairsnucleic acid single linear DNA (genomic) 13 GGCTCGAGAC TGCTAATAGTTCTGCGCAT 29 28 base pairs nucleic acid single linear DNA (genomic) 14GCCATATGAA AAAAACATTA TTAATAGC 28 29 base pairs nucleic acid singlelinear DNA (genomic) 15 GGCTCGAGAC GAAAAATAAC TTCGCTGAA 29 29 base pairsnucleic acid single linear DNA (genomic) 16 GCCATATGAA GTGTATATTATTTAAATGG 29 30 base pairs nucleic acid single linear DNA (genomic) 17GGCTCGAGTT TACCCGTTGT ATATAAAAAC 30 26 base pairs nucleic acid singlelinear DNA (genomic) 18 CGCATATGAA GAAGATGTTT ATGGCG 26 29 base pairsnucleic acid single linear DNA (genomic) 19 GGCTCGAGGT CATTATTAAACTGCACTTC 29 969 base pairs nucleic acid single linear DNA (genomic) CDS1..969 20 ATG AAA ATA ATT ATT TTT AGA GTG CTA ACT TTT TTC TTT GTT ATCTTT 48 Met Lys Ile Ile Ile Phe Arg Val Leu Thr Phe Phe Phe Val Ile Phe 15 10 15 TCA GTT AAT GTG GTG GCG AAG GAA TTT ACC TTA GAC TTC TCG ACT GCA96 Ser Val Asn Val Val Ala Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala 20 2530 AAG ACG TAT GTA GAT TCG CTG AAT GTC ATT CGC TCT GCA ATA GGT ACT 144Lys Thr Tyr Val Asp Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr 35 40 45CCA TTA CAG ACT ATT TCA TCA GGA GGT ACG TCT TTA CTG ATG ATT GAT 192 ProLeu Gln Thr Ile Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp 50 55 60 AGTGGC TCA GGG GAT AAT TTG TTT GCA GTT GAT GTC AGA GGG ATA GAT 240 Ser GlySer Gly Asp Asn Leu Phe Ala Val Asp Val Arg Gly Ile Asp 65 70 75 80 GCAGAG GAA GGG CGG TTT AAT AAT CTA CGG CTT ATT GTT GAA CGA AAT 288 Ala GluGlu Gly Arg Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn 85 90 95 AAT TTATAT GTG ACA GGA TTT GTT AAC AGG ACA AAT AAT GTT TTT TAT 336 Asn Leu TyrVal Thr Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr 100 105 110 CGC TTTGCT GAT TTT TCA CAT GTT ACC TTT CCA GGT ACA ACA GCG GTT 384 Arg Phe AlaAsp Phe Ser His Val Thr Phe Pro Gly Thr Thr Ala Val 115 120 125 ACA TTGTCT GGT GAC AGT AGC TAT ACC ACG TTA CAG CGT GTT GCA GGG 432 Thr Leu SerGly Asp Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly 130 135 140 ATC AGTCGT ACG GGG ATG CAG ATA AAT CGC CAT TCG TTG ACT ACT TCT 480 Ile Ser ArgThr Gly Met Gln Ile Asn Arg His Ser Leu Thr Thr Ser 145 150 155 160 TATCTG GAT TTA ATG TCG CAT AGT GGA ACC TCA CTG ACG CAG TCT GTG 528 Tyr LeuAsp Leu Met Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val 165 170 175 GCAAGA GCG ATG TTA CGG TTT GTT ACT GTG ACA GCT GAA GCT TTA CGT 576 Ala ArgAla Met Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg 180 185 190 TTTCGG CAA ATA CAG AGG GGA TTT CGT ACA ACA CTG GAT GAT CTC AGT 624 Phe ArgGln Ile Gln Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser 195 200 205 GGGCGT TCT TAT GTA ATG ACT GCT GAA GAT GTT GAT CTT ACA TTG AAC 672 Gly ArgSer Tyr Val Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn 210 215 220 TGGGGA AGG TTG AGT AGC GTC CTG CCT GAC TAT CAT GGA CAA GAC TCT 720 Trp GlyArg Leu Ser Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser 225 230 235 240GTT CGT GTA GGA AGA ATT TCT TTT GGA AGC ATT AAT GCA ATT CTG GGA 768 ValArg Val Gly Arg Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly 245 250 255AGC GTG GCA TTA ATA CTG AAT TGT CAT CAT CAT GCA TCG CGA GTT GCC 816 SerVal Ala Leu Ile Leu Asn Cys His His His Ala Ser Arg Val Ala 260 265 270AGA ATG GCA TCT GAT GAG TTT CCT TCT ATG TGT CCG GCA GAT GGA AGA 864 ArgMet Ala Ser Asp Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg 275 280 285GTC CGT GGG ATT ACG CAC AAT AAA ATA TTG TGG GAT TCA TCC ACT CTG 912 ValArg Gly Ile Thr His Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu 290 295 300GGG GCA ATT CTG ATG CGC AGA ACT ATT AGC AGT CTC GAG CAC CAC CAC 960 GlyAla Ile Leu Met Arg Arg Thr Ile Ser Ser Leu Glu His His His 305 310 315320 CAC CAC CAC 969 His His His 323 amino acids amino acid linearprotein 21 Met Lys Ile Ile Ile Phe Arg Val Leu Thr Phe Phe Phe Val IlePhe 1 5 10 15 Ser Val Asn Val Val Ala Lys Glu Phe Thr Leu Asp Phe SerThr Ala 20 25 30 Lys Thr Tyr Val Asp Ser Leu Asn Val Ile Arg Ser Ala IleGly Thr 35 40 45 Pro Leu Gln Thr Ile Ser Ser Gly Gly Thr Ser Leu Leu MetIle Asp 50 55 60 Ser Gly Ser Gly Asp Asn Leu Phe Ala Val Asp Val Arg GlyIle Asp 65 70 75 80 Ala Glu Glu Gly Arg Phe Asn Asn Leu Arg Leu Ile ValGlu Arg Asn 85 90 95 Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr Asn AsnVal Phe Tyr 100 105 110 Arg Phe Ala Asp Phe Ser His Val Thr Phe Pro GlyThr Thr Ala Val 115 120 125 Thr Leu Ser Gly Asp Ser Ser Tyr Thr Thr LeuGln Arg Val Ala Gly 130 135 140 Ile Ser Arg Thr Gly Met Gln Ile Asn ArgHis Ser Leu Thr Thr Ser 145 150 155 160 Tyr Leu Asp Leu Met Ser His SerGly Thr Ser Leu Thr Gln Ser Val 165 170 175 Ala Arg Ala Met Leu Arg PheVal Thr Val Thr Ala Glu Ala Leu Arg 180 185 190 Phe Arg Gln Ile Gln ArgGly Phe Arg Thr Thr Leu Asp Asp Leu Ser 195 200 205 Gly Arg Ser Tyr ValMet Thr Ala Glu Asp Val Asp Leu Thr Leu Asn 210 215 220 Trp Gly Arg LeuSer Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser 225 230 235 240 Val ArgVal Gly Arg Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly 245 250 255 SerVal Ala Leu Ile Leu Asn Cys His His His Ala Ser Arg Val Ala 260 265 270Arg Met Ala Ser Asp Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg 275 280285 Val Arg Gly Ile Thr His Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu 290295 300 Gly Ala Ile Leu Met Arg Arg Thr Ile Ser Ser Leu Glu His His His305 310 315 320 His His His 294 base pairs nucleic acid single linearDNA (genomic) CDS 1..294 22 ATG AAA AAA ACA TTA TTA ATA GCT GCA TCG CTTTCA TTT TTT TCA GCA 48 Met Lys Lys Thr Leu Leu Ile Ala Ala Ser Leu SerPhe Phe Ser Ala 1 5 10 15 AGT GCG CTG GCG ACG CCT GAT TGT GTA ACT GGAAAG GTG GAG TAT ACA 96 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly LysVal Glu Tyr Thr 20 25 30 AAA TAT AAT GAT GAC GAT ACC TTT ACA GTT AAA GTGGGT GAT AAA GAA 144 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val GlyAsp Lys Glu 35 40 45 TTA TTT ACC AAC AGA TGG AAT CTT CAG TCT CTT CTT CTCAGT GCG CAA 192 Leu Phe Thr Asn Arg Trp Asn Leu Gln Ser Leu Leu Leu SerAla Gln 50 55 60 ATT ACG GGG ATG ACT GTA ACC ATT AAA ACT AAT GCC TGT CATAAT GGA 240 Ile Thr Gly Met Thr Val Thr Ile Lys Thr Asn Ala Cys His AsnGly 65 70 75 80 GGG GGA TTC AGC GAA GTT ATT TTT CGT CTC GAG CAC CAC CACCAC CAC 288 Gly Gly Phe Ser Glu Val Ile Phe Arg Leu Glu His His His HisHis 85 90 95 CAC TGA 294 His * 98 amino acids amino acid linear protein23 Met Lys Lys Thr Leu Leu Ile Ala Ala Ser Leu Ser Phe Phe Ser Ala 1 510 15 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr 2025 30 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu 3540 45 Leu Phe Thr Asn Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Ala Gln 5055 60 Ile Thr Gly Met Thr Val Thr Ile Lys Thr Asn Ala Cys His Asn Gly 6570 75 80 Gly Gly Phe Ser Glu Val Ile Phe Arg Leu Glu His His His His His85 90 95 His * 981 base pairs nucleic acid single linear DNA (genomic)CDS 1..981 24 ATG AAG TGT ATA TTA TTT AAA TGG GTA CTG TGC CTG TTA CTGGGT TTT 48 Met Lys Cys Ile Leu Phe Lys Trp Val Leu Cys Leu Leu Leu GlyPhe 1 5 10 15 TCT TCG GTA TCC TAT TCC CGG GAG TTT ACG ATA GAC TTT TCGACC CAA 96 Ser Ser Val Ser Tyr Ser Arg Glu Phe Thr Ile Asp Phe Ser ThrGln 20 25 30 CAA AGT TAT GTC TCT TCG TTA AAT AGT ATA CGG ACA GAG ATA TCGACC 144 Gln Ser Tyr Val Ser Ser Leu Asn Ser Ile Arg Thr Glu Ile Ser Thr35 40 45 CCT CTT GAA CAT ATA TCT CAG GGG ACC ACA TCG GTG TCT GTT ATT AAC192 Pro Leu Glu His Ile Ser Gln Gly Thr Thr Ser Val Ser Val Ile Asn 5055 60 CAC ACC CAC GGC AGT TAT TTT GCT GTG GAT ATA CGA GGG CTT GAT GTC240 His Thr His Gly Ser Tyr Phe Ala Val Asp Ile Arg Gly Leu Asp Val 6570 75 80 TAT CAG GCG CGT TTT GAC CAT CTT CGT CTG ATT ATT GAG CAA AAT AAT288 Tyr Gln Ala Arg Phe Asp His Leu Arg Leu Ile Ile Glu Gln Asn Asn 8590 95 TTA TAT GTG GCA GGG TTC GTT AAT ACG GCA ACA AAT ACT TTC TAC CGT336 Leu Tyr Val Ala Gly Phe Val Asn Thr Ala Thr Asn Thr Phe Tyr Arg 100105 110 TTT TCA GAT TTT ACA CAT ATA TCA GTG CCC GGT GTG ACA ACG GTT TCC384 Phe Ser Asp Phe Thr His Ile Ser Val Pro Gly Val Thr Thr Val Ser 115120 125 ATG ACA ACG GAC AGC AGT TAT ACC ACT CTG CAA CGT GTC GCA GCG CTG432 Met Thr Thr Asp Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Ala Leu 130135 140 GAA CGT TCC GGA ATG CAA ATC AGT CGT CAC TCA CTG GTT TCA TCA TAT480 Glu Arg Ser Gly Met Gln Ile Ser Arg His Ser Leu Val Ser Ser Tyr 145150 155 160 CTG GCG TTA ATG GAG TTC AGT GGT AAT ACA ATG ACC AGA GAT GCATCC 528 Leu Ala Leu Met Glu Phe Ser Gly Asn Thr Met Thr Arg Asp Ala Ser165 170 175 AGA GCA GTT CTG CGT TTT GTC ACT GTC ACA GCA GAA GCC TTA CGCTTC 576 Arg Ala Val Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe180 185 190 AGG CAG ATA CAG AGA GAA TTT CGT CAG GCA CTG TCT GAA ACT GCTCCT 624 Arg Gln Ile Gln Arg Glu Phe Arg Gln Ala Leu Ser Glu Thr Ala Pro195 200 205 GTG TAT ACG ATG ACG CCG GGA GAC GTG GAC CTC ACT CTG AAC TGGGGG 672 Val Tyr Thr Met Thr Pro Gly Asp Val Asp Leu Thr Leu Asn Trp Gly210 215 220 CGA ATC AGC AAT GTG CTT CCG GAG TAT CGG GGA GAG GAT GGT GTCAGA 720 Arg Ile Ser Asn Val Leu Pro Glu Tyr Arg Gly Glu Asp Gly Val Arg225 230 235 240 GTG GGG AGA ATA TCC TTT AAT AAT ATA TCA GCG ATA CTG GGGACT GTG 768 Val Gly Arg Ile Ser Phe Asn Asn Ile Ser Ala Ile Leu Gly ThrVal 245 250 255 GCC GTT ATA CTG AAT TGC CAT CAT CAG GGG GCG CGT TCT GTTCGC GCC 816 Ala Val Ile Leu Asn Cys His His Gln Gly Ala Arg Ser Val ArgAla 260 265 270 GTG AAT GAA GAG AGT CAA CCA GAA TGT CAG ATA ACT GGC GACAGG CCT 864 Val Asn Glu Glu Ser Gln Pro Glu Cys Gln Ile Thr Gly Asp ArgPro 275 280 285 GTT ATA AAA ATA AAC AAT ACA TTA TGG GAA AGT AAT ACA GCTGCA GCG 912 Val Ile Lys Ile Asn Asn Thr Leu Trp Glu Ser Asn Thr Ala AlaAla 290 295 300 TTT CTG AAC AGA AAG TCA CAG TTT TTA TAT ACA ACG GGT AAACTC GAG 960 Phe Leu Asn Arg Lys Ser Gln Phe Leu Tyr Thr Thr Gly Lys LeuGlu 305 310 315 320 CAC CAC CAC CAC CAC CAC TGA 981 His His His His HisHis * 325 327 amino acids amino acid linear protein 25 Met Lys Cys IleLeu Phe Lys Trp Val Leu Cys Leu Leu Leu Gly Phe 1 5 10 15 Ser Ser ValSer Tyr Ser Arg Glu Phe Thr Ile Asp Phe Ser Thr Gln 20 25 30 Gln Ser TyrVal Ser Ser Leu Asn Ser Ile Arg Thr Glu Ile Ser Thr 35 40 45 Pro Leu GluHis Ile Ser Gln Gly Thr Thr Ser Val Ser Val Ile Asn 50 55 60 His Thr HisGly Ser Tyr Phe Ala Val Asp Ile Arg Gly Leu Asp Val 65 70 75 80 Tyr GlnAla Arg Phe Asp His Leu Arg Leu Ile Ile Glu Gln Asn Asn 85 90 95 Leu TyrVal Ala Gly Phe Val Asn Thr Ala Thr Asn Thr Phe Tyr Arg 100 105 110 PheSer Asp Phe Thr His Ile Ser Val Pro Gly Val Thr Thr Val Ser 115 120 125Met Thr Thr Asp Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Ala Leu 130 135140 Glu Arg Ser Gly Met Gln Ile Ser Arg His Ser Leu Val Ser Ser Tyr 145150 155 160 Leu Ala Leu Met Glu Phe Ser Gly Asn Thr Met Thr Arg Asp AlaSer 165 170 175 Arg Ala Val Leu Arg Phe Val Thr Val Thr Ala Glu Ala LeuArg Phe 180 185 190 Arg Gln Ile Gln Arg Glu Phe Arg Gln Ala Leu Ser GluThr Ala Pro 195 200 205 Val Tyr Thr Met Thr Pro Gly Asp Val Asp Leu ThrLeu Asn Trp Gly 210 215 220 Arg Ile Ser Asn Val Leu Pro Glu Tyr Arg GlyGlu Asp Gly Val Arg 225 230 235 240 Val Gly Arg Ile Ser Phe Asn Asn IleSer Ala Ile Leu Gly Thr Val 245 250 255 Ala Val Ile Leu Asn Cys His HisGln Gly Ala Arg Ser Val Arg Ala 260 265 270 Val Asn Glu Glu Ser Gln ProGlu Cys Gln Ile Thr Gly Asp Arg Pro 275 280 285 Val Ile Lys Ile Asn AsnThr Leu Trp Glu Ser Asn Thr Ala Ala Ala 290 295 300 Phe Leu Asn Arg LysSer Gln Phe Leu Tyr Thr Thr Gly Lys Leu Glu 305 310 315 320 His His HisHis His His * 325 294 base pairs nucleic acid single linear DNA(genomic) CDS 1..294 26 ATG AAG AAG ATG TTT ATG GCG GTT TTA TTT GCA TTAGCT TCT GTT AAT 48 Met Lys Lys Met Phe Met Ala Val Leu Phe Ala Leu AlaSer Val Asn 1 5 10 15 GCA ATG GCG GCG GAT TGT GCT AAA GGT AAA ATT GAGTTT TCC AAG TAT 96 Ala Met Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu PheSer Lys Tyr 20 25 30 AAT GAG GAT GAC ACA TTT ACA GTG AAG GTT GAC GGG AAAGAA TAC TGG 144 Asn Glu Asp Asp Thr Phe Thr Val Lys Val Asp Gly Lys GluTyr Trp 35 40 45 ACC AGT CGC TGG AAT CTG CAA CCG TTA CTG CAA AGT GCT CAGTTG ACA 192 Thr Ser Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln LeuThr 50 55 60 GGA ATG ACT GTC ACA ATC AAA TCC AGT ACC TGT GAA TCA GGC TCCGGA 240 Gly Met Thr Val Thr Ile Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly65 70 75 80 TTT GCT GAA GTG CAG TTT AAT AAT GAC CTC GAG CAC CAC CAC CACCAC 288 Phe Ala Glu Val Gln Phe Asn Asn Asp Leu Glu His His His His His85 90 95 CAC TGA 294 His * 98 amino acids amino acid linear protein 27Met Lys Lys Met Phe Met Ala Val Leu Phe Ala Leu Ala Ser Val Asn 1 5 1015 Ala Met Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr 20 2530 Asn Glu Asp Asp Thr Phe Thr Val Lys Val Asp Gly Lys Glu Tyr Trp 35 4045 Thr Ser Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr 50 5560 Gly Met Thr Val Thr Ile Lys Ser Ser Thr Cys Glu Ser Gly Ser Gly 65 7075 80 Phe Ala Glu Val Gln Phe Asn Asn Asp Leu Glu His His His His His 8590 95 His * 32 base pairs nucleic acid single linear DNA (genomic) 28CGGAATTCAA GGAATTTACC TTAGACTTCT CG 32 28 base pairs nucleic acid singlelinear DNA (genomic) 29 GGCTCGAGTC AACTGCTAAT AGTTCTGC 28 32 base pairsnucleic acid single linear DNA (genomic) 30 CGGAATTCCG GGAGTTTACGATAGACTTTT CG 32 29 base pairs nucleic acid single linear DNA (genomic)31 GGCTCGAGTT ATTTACCCGT TGTATATAA 29 2127 base pairs nucleic acidsingle linear DNA (genomic) CDS 1..2127 32 ATG AAA ATA AAA ACA GGT GCACGC ATC CTC GCA TTA TCC GCA TTA ACG 48 Met Lys Ile Lys Thr Gly Ala ArgIle Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 ACG ATG ATG TTT TCC GCC TCGGCT CTC GCC AAA ATC GAA GAA GGT AAA 96 Thr Met Met Phe Ser Ala Ser AlaLeu Ala Lys Ile Glu Glu Gly Lys 20 25 30 CTG GTA ATC TGG ATT AAC GGC GATAAA GGC TAT AAC GGT CTC GCT GAA 144 Leu Val Ile Trp Ile Asn Gly Asp LysGly Tyr Asn Gly Leu Ala Glu 35 40 45 GTC GGT AAG AAA TTC GAG AAA GAT ACCGGA ATT AAA GTC ACC GTT GAG 192 Val Gly Lys Lys Phe Glu Lys Asp Thr GlyIle Lys Val Thr Val Glu 50 55 60 CAT CCG GAT AAA CTG GAA GAG AAA TTC CCACAG GTT GCG GCA ACT GGC 240 His Pro Asp Lys Leu Glu Glu Lys Phe Pro GlnVal Ala Ala Thr Gly 65 70 75 80 GAT GGC CCT GAC ATT ATC TTC TGG GCA CACGAC CGC TTT GGT GGC TAC 288 Asp Gly Pro Asp Ile Ile Phe Trp Ala His AspArg Phe Gly Gly Tyr 85 90 95 GCT CAA TCT GGC CTG TTG GCT GAA ATC ACC CCGGAC AAA GCG TTC CAG 336 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro AspLys Ala Phe Gln 100 105 110 GAC AAG CTG TAT CCG TTT ACC TGG GAT GCC GTACGT TAC AAC GGC AAG 384 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val ArgTyr Asn Gly Lys 115 120 125 CTG ATT GCT TAC CCG ATC GCT GTT GAA GCG TTATCG CTG ATT TAT AAC 432 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu SerLeu Ile Tyr Asn 130 135 140 AAA GAT CTG CTG CCG AAC CCG CCA AAA ACC TGGGAA GAG ATC CCG GCG 480 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp GluGlu Ile Pro Ala 145 150 155 160 CTG GAT AAA GAA CTG AAA GCG AAA GGT AAGAGC GCG CTG ATG TTC AAC 528 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys SerAla Leu Met Phe Asn 165 170 175 CTG CAA GAA CCG TAC TTC ACC TGG CCG CTGATT GCT GCT GAC GGG GGT 576 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu IleAla Ala Asp Gly Gly 180 185 190 TAT GCG TTC AAG TAT GAA AAC GGC AAG TACGAC ATT AAA GAC GTG GGC 624 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr AspIle Lys Asp Val Gly 195 200 205 GTG GAT AAC GCT GGC GCG AAA GCG GGT CTGACC TTC CTG GTT GAC CTG 672 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu ThrPhe Leu Val Asp Leu 210 215 220 ATT AAA AAC AAA CAC ATG AAT GCA GAC ACCGAT TAC TCC ATC GCA GAA 720 Ile Lys Asn Lys His Met Asn Ala Asp Thr AspTyr Ser Ile Ala Glu 225 230 235 240 GCT GCC TTT AAT AAA GGC GAA ACA GCGATG ACC ATC AAC GGC CCG TGG 768 Ala Ala Phe Asn Lys Gly Glu Thr Ala MetThr Ile Asn Gly Pro Trp 245 250 255 GCA TGG TCC AAC ATC GAC ACC AGC AAAGTG AAT TAT GGT GTA ACG GTA 816 Ala Trp Ser Asn Ile Asp Thr Ser Lys ValAsn Tyr Gly Val Thr Val 260 265 270 CTG CCG ACC TTC AAG GGT CAA CCA TCCAAA CCG TTC GTT GGC GTG CTG 864 Leu Pro Thr Phe Lys Gly Gln Pro Ser LysPro Phe Val Gly Val Leu 275 280 285 AGC GCA GGT ATT AAC GCC GCC AGT CCGAAC AAA GAG CTG GCG AAA GAG 912 Ser Ala Gly Ile Asn Ala Ala Ser Pro AsnLys Glu Leu Ala Lys Glu 290 295 300 TTC CTC GAA AAC TAT CTG CTG ACT GATGAA GGT CTG GAA GCG GTT AAT 960 Phe Leu Glu Asn Tyr Leu Leu Thr Asp GluGly Leu Glu Ala Val Asn 305 310 315 320 AAA GAC AAA CCG CTG GGT GCC GTAGCG CTG AAG TCT TAC GAG GAA GAG 1008 Lys Asp Lys Pro Leu Gly Ala Val AlaLeu Lys Ser Tyr Glu Glu Glu 325 330 335 TTG GCG AAA GAT CCA CGT ATT GCCGCC ACC ATG GAA AAC GCC CAG AAA 1056 Leu Ala Lys Asp Pro Arg Ile Ala AlaThr Met Glu Asn Ala Gln Lys 340 345 350 GGT GAA ATC ATG CCG AAC ATC CCGCAG ATG TCC GCT TTC TGG TAT GCC 1104 Gly Glu Ile Met Pro Asn Ile Pro GlnMet Ser Ala Phe Trp Tyr Ala 355 360 365 GTG CGT ACT GCG GTG ATC AAC GCCGCC AGC GGT CGT CAG ACT GTC GAT 1152 Val Arg Thr Ala Val Ile Asn Ala AlaSer Gly Arg Gln Thr Val Asp 370 375 380 GAA GCC CTG AAA GAC GCG CAG ACTTCG AGC TCG AAC AAC AAC AAC AAT 1200 Glu Ala Leu Lys Asp Ala Gln Thr SerSer Ser Asn Asn Asn Asn Asn 385 390 395 400 AAC AAT AAC AAC AAC CTC GGGATC GAG GGA AGG ATT TCA GAA TTC AAG 1248 Asn Asn Asn Asn Asn Leu Gly IleGlu Gly Arg Ile Ser Glu Phe Lys 405 410 415 GAA TTT ACC TTA GAC TTC TCGACT GCA AAG ACG TAT GTA GAT TCG CTG 1296 Glu Phe Thr Leu Asp Phe Ser ThrAla Lys Thr Tyr Val Asp Ser Leu 420 425 430 AAT GTC ATT CGC TCT GCA ATAGGT ACT CCA TTA CAG ACT ATT TCA TCA 1344 Asn Val Ile Arg Ser Ala Ile GlyThr Pro Leu Gln Thr Ile Ser Ser 435 440 445 GGA GGT ACG TCT TTA CTG ATGATT GAT AGT GGC TCA GGG GAT AAT TTG 1392 Gly Gly Thr Ser Leu Leu Met IleAsp Ser Gly Ser Gly Asp Asn Leu 450 455 460 TTT GCA GTT GAT GTC AGA GGGATA GAT GCA GAG GAA GGG CGG TTT AAT 1440 Phe Ala Val Asp Val Arg Gly IleAsp Ala Glu Glu Gly Arg Phe Asn 465 470 475 480 AAT CTA CGG CTT ATT GTTGAA CGA AAT AAT TTA TAT GTG ACA GGA TTT 1488 Asn Leu Arg Leu Ile Val GluArg Asn Asn Leu Tyr Val Thr Gly Phe 485 490 495 GTT AAC AGG ACA AAT AATGTT TTT TAT CGC TTT GCT GAT TTT TCA CAT 1536 Val Asn Arg Thr Asn Asn ValPhe Tyr Arg Phe Ala Asp Phe Ser His 500 505 510 GTT ACC TTT CCA GGT ACAACA GCG GTT ACA TTG TCT GGT GAC AGT AGC 1584 Val Thr Phe Pro Gly Thr ThrAla Val Thr Leu Ser Gly Asp Ser Ser 515 520 525 TAT ACC ACG TTA CAG CGTGTT GCA GGG ATC AGT CGT ACG GGG ATG CAG 1632 Tyr Thr Thr Leu Gln Arg ValAla Gly Ile Ser Arg Thr Gly Met Gln 530 535 540 ATA AAT CGC CAT TCG TTGACT ACT TCT TAT CTG GAT TTA ATG TCG CAT 1680 Ile Asn Arg His Ser Leu ThrThr Ser Tyr Leu Asp Leu Met Ser His 545 550 555 560 AGT GGA ACC TCA CTGACG CAG TCT GTG GCA AGA GCG ATG TTA CGG TTT 1728 Ser Gly Thr Ser Leu ThrGln Ser Val Ala Arg Ala Met Leu Arg Phe 565 570 575 GTT ACT GTG ACA GCTGAA GCT TTA CGT TTT CGG CAA ATA CAG AGG GGA 1776 Val Thr Val Thr Ala GluAla Leu Arg Phe Arg Gln Ile Gln Arg Gly 580 585 590 TTT CGT ACA ACA CTGGAT GAT CTC AGT GGG CGT TCT TAT GTA ATG ACT 1824 Phe Arg Thr Thr Leu AspAsp Leu Ser Gly Arg Ser Tyr Val Met Thr 595 600 605 GCT GAA GAT GTT GATCTT ACA TTG AAC TGG GGA AGG TTG AGT AGC GTC 1872 Ala Glu Asp Val Asp LeuThr Leu Asn Trp Gly Arg Leu Ser Ser Val 610 615 620 CTG CCT GAC TAT CATGGA CAA GAC TCT GTT CGT GTA GGA AGA ATT TCT 1920 Leu Pro Asp Tyr His GlyGln Asp Ser Val Arg Val Gly Arg Ile Ser 625 630 635 640 TTT GGA AGC ATTAAT GCA ATT CTG GGA AGC GTG GCA TTA ATA CTG AAT 1968 Phe Gly Ser Ile AsnAla Ile Leu Gly Ser Val Ala Leu Ile Leu Asn 645 650 655 TGT CAT CAT CATGCA TCG CGA GTT GCC AGA ATG GCA TCT GAT GAG TTT 2016 Cys His His His AlaSer Arg Val Ala Arg Met Ala Ser Asp Glu Phe 660 665 670 CCT TCT ATG TGTCCG GCA GAT GGA AGA GTC CGT GGG ATT ACG CAC AAT 2064 Pro Ser Met Cys ProAla Asp Gly Arg Val Arg Gly Ile Thr His Asn 675 680 685 AAA ATA TTG TGGGAT TCA TCC ACT CTG GGG GCA ATT CTG ATG CGC AGA 2112 Lys Ile Leu Trp AspSer Ser Thr Leu Gly Ala Ile Leu Met Arg Arg 690 695 700 ACT ATT AGC AGTTGA 2127 Thr Ile Ser Ser * 705 709 amino acids amino acid linear protein33 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 510 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 2025 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 3540 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 5055 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 6570 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn GlyLys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu IleTyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu GluIle Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys SerAla Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro LeuIle Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly LysTyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys AlaGly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met AsnAla Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn LysGly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser AsnIle Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro ThrPhe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser AlaGly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 PheLeu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys340 345 350 Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp TyrAla 355 360 365 Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln ThrVal Asp 370 375 380 Glu Ala Leu Lys Asp Ala Gln Thr Ser Ser Ser Asn AsnAsn Asn Asn 385 390 395 400 Asn Asn Asn Asn Asn Leu Gly Ile Glu Gly ArgIle Ser Glu Phe Lys 405 410 415 Glu Phe Thr Leu Asp Phe Ser Thr Ala LysThr Tyr Val Asp Ser Leu 420 425 430 Asn Val Ile Arg Ser Ala Ile Gly ThrPro Leu Gln Thr Ile Ser Ser 435 440 445 Gly Gly Thr Ser Leu Leu Met IleAsp Ser Gly Ser Gly Asp Asn Leu 450 455 460 Phe Ala Val Asp Val Arg GlyIle Asp Ala Glu Glu Gly Arg Phe Asn 465 470 475 480 Asn Leu Arg Leu IleVal Glu Arg Asn Asn Leu Tyr Val Thr Gly Phe 485 490 495 Val Asn Arg ThrAsn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser His 500 505 510 Val Thr PhePro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser Ser 515 520 525 Tyr ThrThr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met Gln 530 535 540 IleAsn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser His 545 550 555560 Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg Phe 565570 575 Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg Gly580 585 590 Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val MetThr 595 600 605 Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu SerSer Val 610 615 620 Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val GlyArg Ile Ser 625 630 635 640 Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser ValAla Leu Ile Leu Asn 645 650 655 Cys His His His Ala Ser Arg Val Ala ArgMet Ala Ser Asp Glu Phe 660 665 670 Pro Ser Met Cys Pro Ala Asp Gly ArgVal Arg Gly Ile Thr His Asn 675 680 685 Lys Ile Leu Trp Asp Ser Ser ThrLeu Gly Ala Ile Leu Met Arg Arg 690 695 700 Thr Ile Ser Ser * 705 2136base pairs nucleic acid single linear DNA (genomic) CDS 1..2136 34 ATGAAA ATA AAA ACA GGT GCA CGC ATC CTC GCA TTA TCC GCA TTA ACG 48 Met LysIle Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 ACGATG ATG TTT TCC GCC TCG GCT CTC GCC AAA ATC GAA GAA GGT AAA 96 Thr MetMet Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 CTG GTAATC TGG ATT AAC GGC GAT AAA GGC TAT AAC GGT CTC GCT GAA 144 Leu Val IleTrp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 GTC GGT AAGAAA TTC GAG AAA GAT ACC GGA ATT AAA GTC ACC GTT GAG 192 Val Gly Lys LysPhe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 CAT CCG GAT AAACTG GAA GAG AAA TTC CCA CAG GTT GCG GCA ACT GGC 240 His Pro Asp Lys LeuGlu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 GAT GGC CCT GACATT ATC TTC TGG GCA CAC GAC CGC TTT GGT GGC TAC 288 Asp Gly Pro Asp IleIle Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 GCT CAA TCT GGC CTGTTG GCT GAA ATC ACC CCG GAC AAA GCG TTC CAG 336 Ala Gln Ser Gly Leu LeuAla Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 GAC AAG CTG TAT CCGTTT ACC TGG GAT GCC GTA CGT TAC AAC GGC AAG 384 Asp Lys Leu Tyr Pro PheThr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 CTG ATT GCT TAC CCGATC GCT GTT GAA GCG TTA TCG CTG ATT TAT AAC 432 Leu Ile Ala Tyr Pro IleAla Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 AAA GAT CTG CTG CCGAAC CCG CCA AAA ACC TGG GAA GAG ATC CCG GCG 480 Lys Asp Leu Leu Pro AsnPro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 CTG GAT AAA GAACTG AAA GCG AAA GGT AAG AGC GCG CTG ATG TTC AAC 528 Leu Asp Lys Glu LeuLys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 CTG CAA GAA CCGTAC TTC ACC TGG CCG CTG ATT GCT GCT GAC GGG GGT 576 Leu Gln Glu Pro TyrPhe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 TAT GCG TTC AAGTAT GAA AAC GGC AAG TAC GAC ATT AAA GAC GTG GGC 624 Tyr Ala Phe Lys TyrGlu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 GTG GAT AAC GCTGGC GCG AAA GCG GGT CTG ACC TTC CTG GTT GAC CTG 672 Val Asp Asn Ala GlyAla Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 ATT AAA AAC AAACAC ATG AAT GCA GAC ACC GAT TAC TCC ATC GCA GAA 720 Ile Lys Asn Lys HisMet Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 GCT GCC TTTAAT AAA GGC GAA ACA GCG ATG ACC ATC AAC GGC CCG TGG 768 Ala Ala Phe AsnLys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 GCA TGG TCCAAC ATC GAC ACC AGC AAA GTG AAT TAT GGT GTA ACG GTA 816 Ala Trp Ser AsnIle Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 CTG CCG ACCTTC AAG GGT CAA CCA TCC AAA CCG TTC GTT GGC GTG CTG 864 Leu Pro Thr PheLys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 AGC GCA GGTATT AAC GCC GCC AGT CCG AAC AAA GAG CTG GCG AAA GAG 912 Ser Ala Gly IleAsn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 TTC CTC GAAAAC TAT CTG CTG ACT GAT GAA GGT CTG GAA GCG GTT AAT 960 Phe Leu Glu AsnTyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 AAA GACAAA CCG CTG GGT GCC GTA GCG CTG AAG TCT TAC GAG GAA GAG 1008 Lys Asp LysPro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 TTG GCGAAA GAT CCA CGT ATT GCC GCC ACC ATG GAA AAC GCC CAG AAA 1056 Leu Ala LysAsp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys 340 345 350 GGT GAAATC ATG CCG AAC ATC CCG CAG ATG TCC GCT TTC TGG TAT GCC 1104 Gly Glu IleMet Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala 355 360 365 GTG CGTACT GCG GTG ATC AAC GCC GCC AGC GGT CGT CAG ACT GTC GAT 1152 Val Arg ThrAla Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp 370 375 380 GAA GCCCTG AAA GAC GCG CAG ACT TCG AGC TCG AAC AAC AAC AAC AAT 1200 Glu Ala LeuLys Asp Ala Gln Thr Ser Ser Ser Asn Asn Asn Asn Asn 385 390 395 400 AACAAT AAC AAC AAC CTC GGG ATC GAG GGA AGG ATT TCA GAA TTC CGG 1248 Asn AsnAsn Asn Asn Leu Gly Ile Glu Gly Arg Ile Ser Glu Phe Arg 405 410 415 GAGTTT ACG ATA GAC TTT TCG ACC CAA CAA AGT TAT GTC TCT TCG TTA 1296 Glu PheThr Ile Asp Phe Ser Thr Gln Gln Ser Tyr Val Ser Ser Leu 420 425 430 AATAGT ATA CGG ACA GAG ATA TCG ACC CCT CTT GAA CAT ATA TCT CAG 1344 Asn SerIle Arg Thr Glu Ile Ser Thr Pro Leu Glu His Ile Ser Gln 435 440 445 GGGACC ACA TCG GTG TCT GTT ATT AAC CAC ACC CAC GGC AGT TAT TTT 1392 Gly ThrThr Ser Val Ser Val Ile Asn His Thr His Gly Ser Tyr Phe 450 455 460 GCTGTG GAT ATA CGA GGG CTT GAT GTC TAT CAG GCG CGT TTT GAC CAT 1440 Ala ValAsp Ile Arg Gly Leu Asp Val Tyr Gln Ala Arg Phe Asp His 465 470 475 480CTT CGT CTG ATT ATT GAG CAA AAT AAT TTA TAT GTG GCA GGG TTC GTT 1488 LeuArg Leu Ile Ile Glu Gln Asn Asn Leu Tyr Val Ala Gly Phe Val 485 490 495AAT ACG GCA ACA AAT ACT TTC TAC CGT TTT TCA GAT TTT ACA CAT ATA 1536 AsnThr Ala Thr Asn Thr Phe Tyr Arg Phe Ser Asp Phe Thr His Ile 500 505 510TCA GTG CCC GGT GTG ACA ACG GTT TCC ATG ACA ACG GAC AGC AGT TAT 1584 SerVal Pro Gly Val Thr Thr Val Ser Met Thr Thr Asp Ser Ser Tyr 515 520 525ACC ACT CTG CAA CGT GTC GCA GCG CTG GAA CGT TCC GGA ATG CAA ATC 1632 ThrThr Leu Gln Arg Val Ala Ala Leu Glu Arg Ser Gly Met Gln Ile 530 535 540AGT CGT CAC TCA CTG GTT TCA TCA TAT CTG GCG TTA ATG GAG TTC AGT 1680 SerArg His Ser Leu Val Ser Ser Tyr Leu Ala Leu Met Glu Phe Ser 545 550 555560 GGT AAT ACA ATG ACC AGA GAT GCA TCC AGA GCA GTT CTG CGT TTT GTC 1728Gly Asn Thr Met Thr Arg Asp Ala Ser Arg Ala Val Leu Arg Phe Val 565 570575 ACT GTC ACA GCA GAA GCC TTA CGC TTC AGG CAG ATA CAG AGA GAA TTT 1776Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg Glu Phe 580 585590 CGT CAG GCA CTG TCT GAA ACT GCT CCT GTG TAT ACG ATG ACG CCG GGA 1824Arg Gln Ala Leu Ser Glu Thr Ala Pro Val Tyr Thr Met Thr Pro Gly 595 600605 GAC GTG GAC CTC ACT CTG AAC TGG GGG CGA ATC AGC AAT GTG CTT CCG 1872Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Ile Ser Asn Val Leu Pro 610 615620 GAG TAT CGG GGA GAG GAT GGT GTC AGA GTG GGG AGA ATA TCC TTT AAT 1920Glu Tyr Arg Gly Glu Asp Gly Val Arg Val Gly Arg Ile Ser Phe Asn 625 630635 640 AAT ATA TCA GCG ATA CTG GGG ACT GTG GCC GTT ATA CTG AAT TGC CAT1968 Asn Ile Ser Ala Ile Leu Gly Thr Val Ala Val Ile Leu Asn Cys His 645650 655 CAT CAG GGG GCG CGT TCT GTT CGC GCC GTG AAT GAA GAG AGT CAA CCA2016 His Gln Gly Ala Arg Ser Val Arg Ala Val Asn Glu Glu Ser Gln Pro 660665 670 GAA TGT CAG ATA ACT GGC GAC AGG CCT GTT ATA AAA ATA AAC AAT ACA2064 Glu Cys Gln Ile Thr Gly Asp Arg Pro Val Ile Lys Ile Asn Asn Thr 675680 685 TTA TGG GAA AGT AAT ACA GCT GCA GCG TTT CTG AAC AGA AAG TCA CAG2112 Leu Trp Glu Ser Asn Thr Ala Ala Ala Phe Leu Asn Arg Lys Ser Gln 690695 700 TTT TTA TAT ACA ACG GGT AAA TAA 2136 Phe Leu Tyr Thr Thr GlyLys * 705 710 712 amino acids amino acid linear protein 35 Met Lys IleLys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr MetMet Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu ValIle Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val GlyLys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His ProAsp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 AspGly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 AlaGln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu MetPhe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala AlaAsp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp IleLys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu ThrPhe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp ThrAsp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu ThrAla Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp ThrSer Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys GlyGln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile AsnAla Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu AsnTyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys AspLys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 LeuAla Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys 340 345 350Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala 355 360365 Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp 370375 380 Glu Ala Leu Lys Asp Ala Gln Thr Ser Ser Ser Asn Asn Asn Asn Asn385 390 395 400 Asn Asn Asn Asn Asn Leu Gly Ile Glu Gly Arg Ile Ser GluPhe Arg 405 410 415 Glu Phe Thr Ile Asp Phe Ser Thr Gln Gln Ser Tyr ValSer Ser Leu 420 425 430 Asn Ser Ile Arg Thr Glu Ile Ser Thr Pro Leu GluHis Ile Ser Gln 435 440 445 Gly Thr Thr Ser Val Ser Val Ile Asn His ThrHis Gly Ser Tyr Phe 450 455 460 Ala Val Asp Ile Arg Gly Leu Asp Val TyrGln Ala Arg Phe Asp His 465 470 475 480 Leu Arg Leu Ile Ile Glu Gln AsnAsn Leu Tyr Val Ala Gly Phe Val 485 490 495 Asn Thr Ala Thr Asn Thr PheTyr Arg Phe Ser Asp Phe Thr His Ile 500 505 510 Ser Val Pro Gly Val ThrThr Val Ser Met Thr Thr Asp Ser Ser Tyr 515 520 525 Thr Thr Leu Gln ArgVal Ala Ala Leu Glu Arg Ser Gly Met Gln Ile 530 535 540 Ser Arg His SerLeu Val Ser Ser Tyr Leu Ala Leu Met Glu Phe Ser 545 550 555 560 Gly AsnThr Met Thr Arg Asp Ala Ser Arg Ala Val Leu Arg Phe Val 565 570 575 ThrVal Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg Glu Phe 580 585 590Arg Gln Ala Leu Ser Glu Thr Ala Pro Val Tyr Thr Met Thr Pro Gly 595 600605 Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Ile Ser Asn Val Leu Pro 610615 620 Glu Tyr Arg Gly Glu Asp Gly Val Arg Val Gly Arg Ile Ser Phe Asn625 630 635 640 Asn Ile Ser Ala Ile Leu Gly Thr Val Ala Val Ile Leu AsnCys His 645 650 655 His Gln Gly Ala Arg Ser Val Arg Ala Val Asn Glu GluSer Gln Pro 660 665 670 Glu Cys Gln Ile Thr Gly Asp Arg Pro Val Ile LysIle Asn Asn Thr 675 680 685 Leu Trp Glu Ser Asn Thr Ala Ala Ala Phe LeuAsn Arg Lys Ser Gln 690 695 700 Phe Leu Tyr Thr Thr Gly Lys * 705 710981 base pairs nucleic acid single linear DNA (genomic) CDS 1..981 36ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT TTC GCT 48 MetLys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15ACC GTT GCG CAA GCT GAC TAC AAG GAC GAC GAT GAC AAG AAG CTT GAA 96 ThrVal Ala Gln Ala Asp Tyr Lys Asp Asp Asp Asp Lys Lys Leu Glu 20 25 30 TTCAAG GAA TTT ACC TTA GAC TTC TCG ACT GCA AAG ACG TAT GTA GAT 144 Phe LysGlu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp 35 40 45 TCG CTGAAT GTC ATT CGC TCT GCA ATA GGT ACT CCA TTA CAG ACT ATT 192 Ser Leu AsnVal Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile 50 55 60 TCA TCA GGAGGT ACG TCT TTA CTG ATG ATT GAT AGT GGC TCA GGG GAT 240 Ser Ser Gly GlyThr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp 65 70 75 80 AAT TTG TTTGCA GTT GAT GTC AGA GGG ATA GAT GCA GAG GAA GGG CGG 288 Asn Leu Phe AlaVal Asp Val Arg Gly Ile Asp Ala Glu Glu Gly Arg 85 90 95 TTT AAT AAT CTACGG CTT ATT GTT GAA CGA AAT AAT TTA TAT GTG ACA 336 Phe Asn Asn Leu ArgLeu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr 100 105 110 GGA TTT GTT AACAGG ACA AAT AAT GTT TTT TAT CGC TTT GCT GAT TTT 384 Gly Phe Val Asn ArgThr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe 115 120 125 TCA CAT GTT ACCTTT CCA GGT ACA ACA GCG GTT ACA TTG TCT GGT GAC 432 Ser His Val Thr PhePro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp 130 135 140 AGT AGC TAT ACCACG TTA CAG CGT GTT GCA GGG ATC AGT CGT ACG GGG 480 Ser Ser Tyr Thr ThrLeu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly 145 150 155 160 ATG CAG ATAAAT CGC CAT TCG TTG ACT ACT TCT TAT CTG GAT TTA ATG 528 Met Gln Ile AsnArg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met 165 170 175 TCG CAT AGTGGA ACC TCA CTG ACG CAG TCT GTG GCA AGA GCG ATG TTA 576 Ser His Ser GlyThr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu 180 185 190 CGG TTT GTTACT GTG ACA GCT GAA GCT TTA CGT TTT CGG CAA ATA CAG 624 Arg Phe Val ThrVal Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln 195 200 205 AGG GGA TTTCGT ACA ACA CTG GAT GAT CTC AGT GGG CGT TCT TAT GTA 672 Arg Gly Phe ArgThr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val 210 215 220 ATG ACT GCTGAA GAT GTT GAT CTT ACA TTG AAC TGG GGA AGG TTG AGT 720 Met Thr Ala GluAsp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser 225 230 235 240 AGC GTCCTG CCT GAC TAT CAT GGA CAA GAC TCT GTT CGT GTA GGA AGA 768 Ser Val LeuPro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg 245 250 255 ATT TCTTTT GGA AGC ATT AAT GCA ATT CTG GGA AGC GTG GCA TTA ATA 816 Ile Ser PheGly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile 260 265 270 CTG AATTGT CAT CAT CAT GCA TCG CGA GTT GCC AGA ATG GCA TCT GAT 864 Leu Asn CysHis His His Ala Ser Arg Val Ala Arg Met Ala Ser Asp 275 280 285 GAG TTTCCT TCT ATG TGT CCG GCA GAT GGA AGA GTC CGT GGG ATT ACG 912 Glu Phe ProSer Met Cys Pro Ala Asp Gly Arg Val Arg Gly Ile Thr 290 295 300 CAC AATAAA ATA TTG TGG GAT TCA TCC ACT CTG GGG GCA ATT CTG ATG 960 His Asn LysIle Leu Trp Asp Ser Ser Thr Leu Gly Ala Ile Leu Met 305 310 315 320 CGCAGA ACT ATT AGC AGT TGA 981 Arg Arg Thr Ile Ser Ser * 325 327 aminoacids amino acid linear protein 37 Met Lys Lys Thr Ala Ile Ala Ile AlaVal Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Asp Tyr LysAsp Asp Asp Asp Lys Lys Leu Glu 20 25 30 Phe Lys Glu Phe Thr Leu Asp PheSer Thr Ala Lys Thr Tyr Val Asp 35 40 45 Ser Leu Asn Val Ile Arg Ser AlaIle Gly Thr Pro Leu Gln Thr Ile 50 55 60 Ser Ser Gly Gly Thr Ser Leu LeuMet Ile Asp Ser Gly Ser Gly Asp 65 70 75 80 Asn Leu Phe Ala Val Asp ValArg Gly Ile Asp Ala Glu Glu Gly Arg 85 90 95 Phe Asn Asn Leu Arg Leu IleVal Glu Arg Asn Asn Leu Tyr Val Thr 100 105 110 Gly Phe Val Asn Arg ThrAsn Asn Val Phe Tyr Arg Phe Ala Asp Phe 115 120 125 Ser His Val Thr PhePro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp 130 135 140 Ser Ser Tyr ThrThr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly 145 150 155 160 Met GlnIle Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met 165 170 175 SerHis Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu 180 185 190Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln 195 200205 Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val 210215 220 Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser225 230 235 240 Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg ValGly Arg 245 250 255 Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser ValAla Leu Ile 260 265 270 Leu Asn Cys His His His Ala Ser Arg Val Ala ArgMet Ala Ser Asp 275 280 285 Glu Phe Pro Ser Met Cys Pro Ala Asp Gly ArgVal Arg Gly Ile Thr 290 295 300 His Asn Lys Ile Leu Trp Asp Ser Ser ThrLeu Gly Ala Ile Leu Met 305 310 315 320 Arg Arg Thr Ile Ser Ser * 325990 base pairs nucleic acid single linear DNA (genomic) CDS 1..990 38ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT TTC GCT 48 MetLys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15ACC GTT GCG CAA GCT GAC TAC AAG GAC GAC GAT GAC AAG AAG CTT GAA 96 ThrVal Ala Gln Ala Asp Tyr Lys Asp Asp Asp Asp Lys Lys Leu Glu 20 25 30 TTCCGG GAG TTT ACG ATA GAC TTT TCG ACC CAA CAA AGT TAT GTC TCT 144 Phe ArgGlu Phe Thr Ile Asp Phe Ser Thr Gln Gln Ser Tyr Val Ser 35 40 45 TCG TTAAAT AGT ATA CGG ACA GAG ATA TCG ACC CCT CTT GAA CAT ATA 192 Ser Leu AsnSer Ile Arg Thr Glu Ile Ser Thr Pro Leu Glu His Ile 50 55 60 TCT CAG GGGACC ACA TCG GTG TCT GTT ATT AAC CAC ACC CAC GGC AGT 240 Ser Gln Gly ThrThr Ser Val Ser Val Ile Asn His Thr His Gly Ser 65 70 75 80 TAT TTT GCTGTG GAT ATA CGA GGG CTT GAT GTC TAT CAG GCG CGT TTT 288 Tyr Phe Ala ValAsp Ile Arg Gly Leu Asp Val Tyr Gln Ala Arg Phe 85 90 95 GAC CAT CTT CGTCTG ATT ATT GAG CAA AAT AAT TTA TAT GTG GCA GGG 336 Asp His Leu Arg LeuIle Ile Glu Gln Asn Asn Leu Tyr Val Ala Gly 100 105 110 TTC GTT AAT ACGGCA ACA AAT ACT TTC TAC CGT TTT TCA GAT TTT ACA 384 Phe Val Asn Thr AlaThr Asn Thr Phe Tyr Arg Phe Ser Asp Phe Thr 115 120 125 CAT ATA TCA GTGCCC GGT GTG ACA ACG GTT TCC ATG ACA ACG GAC AGC 432 His Ile Ser Val ProGly Val Thr Thr Val Ser Met Thr Thr Asp Ser 130 135 140 AGT TAT ACC ACTCTG CAA CGT GTC GCA GCG CTG GAA CGT TCC GGA ATG 480 Ser Tyr Thr Thr LeuGln Arg Val Ala Ala Leu Glu Arg Ser Gly Met 145 150 155 160 CAA ATC AGTCGT CAC TCA CTG GTT TCA TCA TAT CTG GCG TTA ATG GAG 528 Gln Ile Ser ArgHis Ser Leu Val Ser Ser Tyr Leu Ala Leu Met Glu 165 170 175 TTC AGT GGTAAT ACA ATG ACC AGA GAT GCA TCC AGA GCA GTT CTG CGT 576 Phe Ser Gly AsnThr Met Thr Arg Asp Ala Ser Arg Ala Val Leu Arg 180 185 190 TTT GTC ACTGTC ACA GCA GAA GCC TTA CGC TTC AGG CAG ATA CAG AGA 624 Phe Val Thr ValThr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 195 200 205 GAA TTT CGTCAG GCA CTG TCT GAA ACT GCT CCT GTG TAT ACG ATG ACG 672 Glu Phe Arg GlnAla Leu Ser Glu Thr Ala Pro Val Tyr Thr Met Thr 210 215 220 CCG GGA GACGTG GAC CTC ACT CTG AAC TGG GGG CGA ATC AGC AAT GTG 720 Pro Gly Asp ValAsp Leu Thr Leu Asn Trp Gly Arg Ile Ser Asn Val 225 230 235 240 CTT CCGGAG TAT CGG GGA GAG GAT GGT GTC AGA GTG GGG AGA ATA TCC 768 Leu Pro GluTyr Arg Gly Glu Asp Gly Val Arg Val Gly Arg Ile Ser 245 250 255 TTT AATAAT ATA TCA GCG ATA CTG GGG ACT GTG GCC GTT ATA CTG AAT 816 Phe Asn AsnIle Ser Ala Ile Leu Gly Thr Val Ala Val Ile Leu Asn 260 265 270 TGC CATCAT CAG GGG GCG CGT TCT GTT CGC GCC GTG AAT GAA GAG AGT 864 Cys His HisGln Gly Ala Arg Ser Val Arg Ala Val Asn Glu Glu Ser 275 280 285 CAA CCAGAA TGT CAG ATA ACT GGC GAC AGG CCT GTT ATA AAA ATA AAC 912 Gln Pro GluCys Gln Ile Thr Gly Asp Arg Pro Val Ile Lys Ile Asn 290 295 300 AAT ACATTA TGG GAA AGT AAT ACA GCT GCA GCG TTT CTG AAC AGA AAG 960 Asn Thr LeuTrp Glu Ser Asn Thr Ala Ala Ala Phe Leu Asn Arg Lys 305 310 315 320 TCACAG TTT TTA TAT ACA ACG GGT AAA TAA 990 Ser Gln Phe Leu Tyr Thr Thr GlyLys * 325 330 330 amino acids amino acid linear protein 39 Met Lys LysThr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr ValAla Gln Ala Asp Tyr Lys Asp Asp Asp Asp Lys Lys Leu Glu 20 25 30 Phe ArgGlu Phe Thr Ile Asp Phe Ser Thr Gln Gln Ser Tyr Val Ser 35 40 45 Ser LeuAsn Ser Ile Arg Thr Glu Ile Ser Thr Pro Leu Glu His Ile 50 55 60 Ser GlnGly Thr Thr Ser Val Ser Val Ile Asn His Thr His Gly Ser 65 70 75 80 TyrPhe Ala Val Asp Ile Arg Gly Leu Asp Val Tyr Gln Ala Arg Phe 85 90 95 AspHis Leu Arg Leu Ile Ile Glu Gln Asn Asn Leu Tyr Val Ala Gly 100 105 110Phe Val Asn Thr Ala Thr Asn Thr Phe Tyr Arg Phe Ser Asp Phe Thr 115 120125 His Ile Ser Val Pro Gly Val Thr Thr Val Ser Met Thr Thr Asp Ser 130135 140 Ser Tyr Thr Thr Leu Gln Arg Val Ala Ala Leu Glu Arg Ser Gly Met145 150 155 160 Gln Ile Ser Arg His Ser Leu Val Ser Ser Tyr Leu Ala LeuMet Glu 165 170 175 Phe Ser Gly Asn Thr Met Thr Arg Asp Ala Ser Arg AlaVal Leu Arg 180 185 190 Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe ArgGln Ile Gln Arg 195 200 205 Glu Phe Arg Gln Ala Leu Ser Glu Thr Ala ProVal Tyr Thr Met Thr 210 215 220 Pro Gly Asp Val Asp Leu Thr Leu Asn TrpGly Arg Ile Ser Asn Val 225 230 235 240 Leu Pro Glu Tyr Arg Gly Glu AspGly Val Arg Val Gly Arg Ile Ser 245 250 255 Phe Asn Asn Ile Ser Ala IleLeu Gly Thr Val Ala Val Ile Leu Asn 260 265 270 Cys His His Gln Gly AlaArg Ser Val Arg Ala Val Asn Glu Glu Ser 275 280 285 Gln Pro Glu Cys GlnIle Thr Gly Asp Arg Pro Val Ile Lys Ile Asn 290 295 300 Asn Thr Leu TrpGlu Ser Asn Thr Ala Ala Ala Phe Leu Asn Arg Lys 305 310 315 320 Ser GlnPhe Leu Tyr Thr Thr Gly Lys * 325 330 26 base pairs nucleic acid singlelinear other nucleic acid /desc = “DNA” 40 GCCATATGAA GGAATTTACC TTAGAC26 26 base pairs nucleic acid single linear other nucleic acid /desc =“DNA” 41 GCCATATGCG GGAGTTTACG ATAGAC 26 26 base pairs nucleic acidsingle linear other nucleic acid /desc = “DNA” 42 GCCATATGAC GCCTGATTGTGTAACT 26 25 base pairs nucleic acid single linear other nucleic acid/desc = “DNA” 43 GCCATATGGC GGATTGTGCT AAAGG 25 32 base pairs nucleicacid single linear other nucleic acid /desc = “DNA” 44 GGCTCGAGTCAACGAAAAAT AACTTCGCTG AA 32 32 base pairs nucleic acid single linearother nucleic acid /desc = “DNA” 45 GGCTCGAGTC AGTCATTATT AAACTGCACT TC32 2073 base pairs nucleic acid double linear other nucleic acid /desc =“DNA” CDS 1..2070 46 ATG AAA ATC GAA GAA GGT AAA CTG GTA ATC TGG ATT AACGGC GAT AAA 48 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn GlyAsp Lys 1 5 10 15 GGC TAT AAC GGT CTC GCT GAA GTC GGT AAG AAA TTC GAGAAA GAT ACC 96 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu LysAsp Thr 20 25 30 GGA ATT AAA GTC ACC GTT GAG CAT CCG GAT AAA CTG GAA GAGAAA TTC 144 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu LysPhe 35 40 45 CCA CAG GTT GCG GCA ACT GGC GAT GGC CCT GAC ATT ATC TTC TGGGCA 192 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala50 55 60 CAC GAC CGC TTT GGT GGC TAC GCT CAA TCT GGC CTG TTG GCT GAA ATC240 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 6570 75 80 ACC CCG GAC AAA GCG TTC CAG GAC AAG CTG TAT CCG TTT ACC TGG GAT288 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 8590 95 GCC GTA CGT TAC AAC GGC AAG CTG ATT GCT TAC CCG ATC GCT GTT GAA336 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100105 110 GCG TTA TCG CTG ATT TAT AAC AAA GAT CTG CTG CCG AAC CCG CCA AAA384 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115120 125 ACC TGG GAA GAG ATC CCG GCG CTG GAT AAA GAA CTG AAA GCG AAA GGT432 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130135 140 AAG AGC GCG CTG ATG TTC AAC CTG CAA GAA CCG TAC TTC ACC TGG CCG480 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145150 155 160 CTG ATT GCT GCT GAC GGG GGT TAT GCG TTC AAG TAT GAA AAC GGCAAG 528 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys165 170 175 TAC GAC ATT AAA GAC GTG GGC GTG GAT AAC GCT GGC GCG AAA GCGGGT 576 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly180 185 190 CTG ACC TTC CTG GTT GAC CTG ATT AAA AAC AAA CAC ATG AAT GCAGAC 624 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp195 200 205 ACC GAT TAC TCC ATC GCA GAA GCT GCC TTT AAT AAA GGC GAA ACAGCG 672 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala210 215 220 ATG ACC ATC AAC GGC CCG TGG GCA TGG TCC AAC ATC GAC ACC AGCAAA 720 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys225 230 235 240 GTG AAT TAT GGT GTA ACG GTA CTG CCG ACC TTC AAG GGT CAACCA TCC 768 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln ProSer 245 250 255 AAA CCG TTC GTT GGC GTG CTG AGC GCA GGT ATT AAC GCC GCCAGT CCG 816 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala SerPro 260 265 270 AAC AAA GAG CTG GCA AAA GAG TTC CTC GAA AAC TAT CTG CTGACT GAT 864 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu ThrAsp 275 280 285 GAA GGT CTG GAA GCG GTT AAT AAA GAC AAA CCG CTG GGT GCCGTA GCG 912 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala ValAla 290 295 300 CTG AAG TCT TAC GAG GAA GAG TTG GCG AAA GAT CCA CGT ATTGCC GCC 960 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile AlaAla 305 310 315 320 ACC ATG GAA AAC GCC CAG AAA GGT GAA ATC ATG CCG AACATC CCG CAG 1008 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn IlePro Gln 325 330 335 ATG TCC GCT TTC TGG TAT GCC GTG CGT ACT GCG GTG ATCAAC GCC GCC 1056 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile AsnAla Ala 340 345 350 AGC GGT CGT CAG ACT GTC GAT GAA GCC CTG AAA GAC GCGCAG ACT AAT 1104 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala GlnThr Asn 355 360 365 TCG AGC TCG AAC AAC AAC AAC AAT AAC AAT AAC AAC AACCTC GGG ATC 1152 Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn LeuGly Ile 370 375 380 GAG GGA AGG ATT TCA GAA TTC GGA TCC GCC CCG GAA TTCAAG GAA TTT 1200 Glu Gly Arg Ile Ser Glu Phe Gly Ser Ala Pro Glu Phe LysGlu Phe 385 390 395 400 ACC TTA GAC TTC TCG ACT GCA AAG ACG TAT GTA GATTCG CTG AAT GTC 1248 Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp SerLeu Asn Val 405 410 415 ATT CGC TCT GCA ATA GGT ACT CCA TTA CAG ACT ATTTCA TCA GGA GGT 1296 Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile SerSer Gly Gly 420 425 430 ACG TCT TTA CTG ATG ATT GAT AGT GGC TCA GGG GATAAT TTG TTT GCA 1344 Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp AsnLeu Phe Ala 435 440 445 GTT GAT GTC AGA GGG ATA GAT CCA GAG GAA GGG CGGTTT AAT AAT CTA 1392 Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg PheAsn Asn Leu 450 455 460 CGG CTT ATT GTT GAA CGA AAT AAT TTA TAT GTG ACAGGA TTT GTT AAC 1440 Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr GlyPhe Val Asn 465 470 475 480 AGG ACA AAT AAT GTT TTT TAT CGC TTT GCT GATTTT TCA CAT GTT ACC 1488 Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp PheSer His Val Thr 485 490 495 TTT CCA GGT ACA ACA GCG GTT ACA TTG TCT GGTGAC AGT AGC TAT ACC 1536 Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly AspSer Ser Tyr Thr 500 505 510 ACG TTA CAG CGT GTT GCA GGG ATC AGT CGT ACGGGG ATG CAG ATA AAT 1584 Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr GlyMet Gln Ile Asn 515 520 525 CGC CAT TCG TTG ACT ACT TCT TAT CTG GAT TTAATG TCG CAT AGT GGA 1632 Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu MetSer His Ser Gly 530 535 540 ACC TCA CTG ACG CAG TCT GTG GCA AGA GCG ATGTTA CGG TTT GTT ACT 1680 Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met LeuArg Phe Val Thr 545 550 555 560 GTG ACA GCT GAA GCT TTA CGT TTT CGG CAAATA CAG AGG GGA TTT CGT 1728 Val Thr Ala Glu Ala Leu Arg Phe Arg Gln IleGln Arg Gly Phe Arg 565 570 575 ACA ACA CTG GAT GAT CTC AGT GGG CGT TCTTAT GTA ATG ACT GCT GAA 1776 Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser TyrVal Met Thr Ala Glu 580 585 590 GAT GTT GAT CTT ACA TTG AAC TGG GGA AGGTTG AGT AGC GTC CTG CCT 1824 Asp Val Asp Leu Thr Leu Asn Trp Gly Arg LeuSer Ser Val Leu Pro 595 600 605 GAC TAT CAT GGA CAA GAC TCT GTT CGT GTAGGA AGA ATT TCT TTT GGA 1872 Asp Tyr His Gly Gln Asp Ser Val Arg Val GlyArg Ile Ser Phe Gly 610 615 620 AGC ATT AAT GCA ATT CTG GGA AGC GTG GCATTA ATA CTG AAT TGT CAT 1920 Ser Ile Asn Ala Ile Leu Gly Ser Val Ala LeuIle Leu Asn Cys His 625 630 635 640 CAT CAT GCA TCG CGA GTT GCC AGA ATGGCA TCT GAT GAG TTT CCT TCT 1968 His His Ala Ser Arg Val Ala Arg Met AlaSer Asp Glu Phe Pro Ser 645 650 655 ATG TGT CCG GCA GAT GGA AGA GTC CGTGGG ATT ACG CAC AAT AAA ATA 2016 Met Cys Pro Ala Asp Gly Arg Val Arg GlyIle Thr His Asn Lys Ile 660 665 670 TTG TGG GAT TCA TCC ACT CTG GGG GCAATT CTG ATG CGC AGA ACT ATT 2064 Leu Trp Asp Ser Ser Thr Leu Gly Ala IleLeu Met Arg Arg Thr Ile 675 680 685 AGC AGT TGA 2073 Ser Ser 690 690amino acids amino acid linear protein 47 Met Lys Ile Glu Glu Gly Lys LeuVal Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala GluVal Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val GluHis Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr GlyAsp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly TyrAla Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala PheGln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn GlyLys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu IleTyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu GluIle Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser AlaLeu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 LeuIle Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr SerLys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys GlyGln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile AsnAla Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu AsnTyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp LysPro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu AlaLys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln LysGly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp TyrAla Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln ThrVal Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser AsnAsn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile 370 375 380 Glu Gly ArgIle Ser Glu Phe Gly Ser Ala Pro Glu Phe Lys Glu Phe 385 390 395 400 ThrLeu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser Leu Asn Val 405 410 415Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser Ser Gly Gly 420 425430 Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn Leu Phe Ala 435440 445 Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe Asn Asn Leu450 455 460 Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly Phe ValAsn 465 470 475 480 Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe SerHis Val Thr 485 490 495 Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly AspSer Ser Tyr Thr 500 505 510 Thr Leu Gln Arg Val Ala Gly Ile Ser Arg ThrGly Met Gln Ile Asn 515 520 525 Arg His Ser Leu Thr Thr Ser Tyr Leu AspLeu Met Ser His Ser Gly 530 535 540 Thr Ser Leu Thr Gln Ser Val Ala ArgAla Met Leu Arg Phe Val Thr 545 550 555 560 Val Thr Ala Glu Ala Leu ArgPhe Arg Gln Ile Gln Arg Gly Phe Arg 565 570 575 Thr Thr Leu Asp Asp LeuSer Gly Arg Ser Tyr Val Met Thr Ala Glu 580 585 590 Asp Val Asp Leu ThrLeu Asn Trp Gly Arg Leu Ser Ser Val Leu Pro 595 600 605 Asp Tyr His GlyGln Asp Ser Val Arg Val Gly Arg Ile Ser Phe Gly 610 615 620 Ser Ile AsnAla Ile Leu Gly Ser Val Ala Leu Ile Leu Asn Cys His 625 630 635 640 HisHis Ala Ser Arg Val Ala Arg Met Ala Ser Asp Glu Phe Pro Ser 645 650 655Met Cys Pro Ala Asp Gly Arg Val Arg Gly Ile Thr His Asn Lys Ile 660 665670 Leu Trp Asp Ser Ser Thr Leu Gly Ala Ile Leu Met Arg Arg Thr Ile 675680 685 Ser Ser 690 2085 base pairs nucleic acid double linear othernucleic acid /desc = “DNA” CDS 1..2082 48 ATG AAA ATC GAA GAA GGT AAACTG GTA ATC TGG ATT AAC GGC GAT AAA 48 Met Lys Ile Glu Glu Gly Lys LeuVal Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 GGC TAT AAC GGT CTC GCT GAAGTC GGT AAG AAA TTC GAG AAA GAT ACC 96 Gly Tyr Asn Gly Leu Ala Glu ValGly Lys Lys Phe Glu Lys Asp Thr 20 25 30 GGA ATT AAA GTC ACC GTT GAG CATCCG GAT AAA CTG GAA GAG AAA TTC 144 Gly Ile Lys Val Thr Val Glu His ProAsp Lys Leu Glu Glu Lys Phe 35 40 45 CCA CAG GTT GCG GCA ACT GGC GAT GGCCCT GAC ATT ATC TTC TGG GCA 192 Pro Gln Val Ala Ala Thr Gly Asp Gly ProAsp Ile Ile Phe Trp Ala 50 55 60 CAC GAC CGC TTT GGT GGC TAC GCT CAA TCTGGC CTG TTG GCT GAA ATC 240 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser GlyLeu Leu Ala Glu Ile 65 70 75 80 ACC CCG GAC AAA GCG TTC CAG GAC AAG CTGTAT CCG TTT ACC TGG GAT 288 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu TyrPro Phe Thr Trp Asp 85 90 95 GCC GTA CGT TAC AAC GGC AAG CTG ATT GCT TACCCG ATC GCT GTT GAA 336 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr ProIle Ala Val Glu 100 105 110 GCG TTA TCG CTG ATT TAT AAC AAA GAT CTG CTGCCG AAC CCG CCA AAA 384 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu ProAsn Pro Pro Lys 115 120 125 ACC TGG GAA GAG ATC CCG GCG CTG GAT AAA GAACTG AAA GCG AAA GGT 432 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu LeuLys Ala Lys Gly 130 135 140 AAG AGC GCG CTG ATG TTC AAC CTG CAA GAA CCGTAC TTC ACC TGG CCG 480 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro TyrPhe Thr Trp Pro 145 150 155 160 CTG ATT GCT GCT GAC GGG GGT TAT GCG TTCAAG TAT GAA AAC GGC AAG 528 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe LysTyr Glu Asn Gly Lys 165 170 175 TAC GAC ATT AAA GAC GTG GGC GTG GAT AACGCT GGC GCG AAA GCG GGT 576 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn AlaGly Ala Lys Ala Gly 180 185 190 CTG ACC TTC CTG GTT GAC CTG ATT AAA AACAAA CAC ATG AAT GCA GAC 624 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn LysHis Met Asn Ala Asp 195 200 205 ACC GAT TAC TCC ATC GCA GAA GCT GCC TTTAAT AAA GGC GAA ACA GCG 672 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe AsnLys Gly Glu Thr Ala 210 215 220 ATG ACC ATC AAC GGC CCG TGG GCA TGG TCCAAC ATC GAC ACC AGC AAA 720 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser AsnIle Asp Thr Ser Lys 225 230 235 240 GTG AAT TAT GGT GTA ACG GTA CTG CCGACC TTC AAG GGT CAA CCA TCC 768 Val Asn Tyr Gly Val Thr Val Leu Pro ThrPhe Lys Gly Gln Pro Ser 245 250 255 AAA CCG TTC GTT GGC GTG CTG AGC GCAGGT ATT AAC GCC GCC AGT CCG 816 Lys Pro Phe Val Gly Val Leu Ser Ala GlyIle Asn Ala Ala Ser Pro 260 265 270 AAC AAA GAG CTG GCA AAA GAG TTC CTCGAA AAC TAT CTG CTG ACT GAT 864 Asn Lys Glu Leu Ala Lys Glu Phe Leu GluAsn Tyr Leu Leu Thr Asp 275 280 285 GAA GGT CTG GAA GCG GTT AAT AAA GACAAA CCG CTG GGT GCC GTA GCG 912 Glu Gly Leu Glu Ala Val Asn Lys Asp LysPro Leu Gly Ala Val Ala 290 295 300 CTG AAG TCT TAC GAG GAA GAG TTG GCGAAA GAT CCA CGT ATT GCC GCC 960 Leu Lys Ser Tyr Glu Glu Glu Leu Ala LysAsp Pro Arg Ile Ala Ala 305 310 315 320 ACC ATG GAA AAC GCC CAG AAA GGTGAA ATC ATG CCG AAC ATC CCG CAG 1008 Thr Met Glu Asn Ala Gln Lys Gly GluIle Met Pro Asn Ile Pro Gln 325 330 335 ATG TCC GCT TTC TGG TAT GCC GTGCGT ACT GCG GTG ATC AAC GCC GCC 1056 Met Ser Ala Phe Trp Tyr Ala Val ArgThr Ala Val Ile Asn Ala Ala 340 345 350 AGC GGT CGT CAG ACT GTC GAT GAAGCC CTG AAA GAC GCG CAG ACT AAT 1104 Ser Gly Arg Gln Thr Val Asp Glu AlaLeu Lys Asp Ala Gln Thr Asn 355 360 365 TCG AGC TCG AAC AAC AAC AAC AATAAC AAT AAC AAC AAC CTC GGG ATC 1152 Ser Ser Ser Asn Asn Asn Asn Asn AsnAsn Asn Asn Asn Leu Gly Ile 370 375 380 GAG GGA AGG ATT TCA GAA TTC GGATCC GCC CCG GAA TTC CGG GAG TTT 1200 Glu Gly Arg Ile Ser Glu Phe Gly SerAla Pro Glu Phe Arg Glu Phe 385 390 395 400 ACG ATA GAC TTT TCG ACC CAACAA AGT TAT GTC TCT TCG TTA AAT AGT 1248 Thr Ile Asp Phe Ser Thr Gln GlnSer Tyr Val Ser Ser Leu Asn Ser 405 410 415 ATA CGG ACA GAG ATA TCG ACCCCT CTT GAA CAT ATA TCT CAG GGG ACC 1296 Ile Arg Thr Glu Ile Ser Thr ProLeu Glu His Ile Ser Gln Gly Thr 420 425 430 ACA TCG GTG TCT GTT ATT AACCAC ACC CCA CCG GGC AGT TAT TTT GCT 1344 Thr Ser Val Ser Val Ile Asn HisThr Pro Pro Gly Ser Tyr Phe Ala 435 440 445 GTG GAT ATA CGA GGG CTT GATGTC TAT CAG GCG CGT TTT GAC CAT CTT 1392 Val Asp Ile Arg Gly Leu Asp ValTyr Gln Ala Arg Phe Asp His Leu 450 455 460 CGT CTG ATT ATT GAG CAA AATAAT TTA TAT GTG GCC GGG TTC GTT AAT 1440 Arg Leu Ile Ile Glu Gln Asn AsnLeu Tyr Val Ala Gly Phe Val Asn 465 470 475 480 ACG GCA ACA AAT ACT TTCTAC CGT TTT TCA GAT TTT ACA CAT ATA TCA 1488 Thr Ala Thr Asn Thr Phe TyrArg Phe Ser Asp Phe Thr His Ile Ser 485 490 495 GTG CCC GGT GTG ACA ACGGTT TCC ATG ACA ACG GAC AGC AGT TAT ACC 1536 Val Pro Gly Val Thr Thr ValSer Met Thr Thr Asp Ser Ser Tyr Thr 500 505 510 ACT CTG CAA CGT GTC GCAGCG CTG GAA CGT TCC GGA ATG CAA ATC AGT 1584 Thr Leu Gln Arg Val Ala AlaLeu Glu Arg Ser Gly Met Gln Ile Ser 515 520 525 CGT CAC TCA CTG GTT TCATCA TAT CTG GCG TTA ATG GAG TTC AGT GGT 1632 Arg His Ser Leu Val Ser SerTyr Leu Ala Leu Met Glu Phe Ser Gly 530 535 540 AAT ACA ATG ACC AGA GATGCA TCC AGA GCA GTT CTG CGT TTT GTC ACT 1680 Asn Thr Met Thr Arg Asp AlaSer Arg Ala Val Leu Arg Phe Val Thr 545 550 555 560 GTC ACA GCA GAA GCCTTA CGC TTC AGG CAG ATA CAG AGA GAA TTT CGT 1728 Val Thr Ala Glu Ala LeuArg Phe Arg Gln Ile Gln Arg Glu Phe Arg 565 570 575 CAG GCA CTG TCT GAAACT GCT CCT GTG TAT ACG ATG ACG CCG GGA GAC 1776 Gln Ala Leu Ser Glu ThrAla Pro Val Tyr Thr Met Thr Pro Gly Asp 580 585 590 GTG GAC CTC ACT CTGAAC TGG GGG CGA ATC AGC AAT GTG CTT CCG GAG 1824 Val Asp Leu Thr Leu AsnTrp Gly Arg Ile Ser Asn Val Leu Pro Glu 595 600 605 TAT CGG GGA GAG GATGGT GTC AGA GTG GGG AGA ATA TCC TTT AAT AAT 1872 Tyr Arg Gly Glu Asp GlyVal Arg Val Gly Arg Ile Ser Phe Asn Asn 610 615 620 ATA TCA GCG ATA CTGGGG ACT GTG GCC GTT ATA CTG AAT TGC CAT CAT 1920 Ile Ser Ala Ile Leu GlyThr Val Ala Val Ile Leu Asn Cys His His 625 630 635 640 CAG GGG GCG CGTTCT GTT CGC GCC GTG AAT GAA GAG AGT CAA CCA GAA 1968 Gln Gly Ala Arg SerVal Arg Ala Val Asn Glu Glu Ser Gln Pro Glu 645 650 655 TGT CAG ATA ACTGGC GAC AGG CCT GTT ATA AAA ATA AAC AAT ACA TTA 2016 Cys Gln Ile Thr GlyAsp Arg Pro Val Ile Lys Ile Asn Asn Thr Leu 660 665 670 TGG GAA AGT AATACA GCT GCA GCG TTT CTG AAC AGA AAG TCA CAG TTT 2064 Trp Glu Ser Asn ThrAla Ala Ala Phe Leu Asn Arg Lys Ser Gln Phe 675 680 685 TTA TAT ACA ACGGGT AAA TAA 2085 Leu Tyr Thr Thr Gly Lys 690 694 amino acids amino acidlinear protein 49 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile AsnGly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys PheGlu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys LeuGlu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp IleIle Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly LeuLeu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu TyrPro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala TyrPro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp LeuLeu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu AspLys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn LeuGln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp GlyGly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys AspVal Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe LeuVal Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp TyrSer Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met ThrIle Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala ValAla 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg IleAla Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met ProAsn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr AlaVal Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala LeuLys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Asn Asn Asn Asn Asn AsnAsn Asn Asn Asn Leu Gly Ile 370 375 380 Glu Gly Arg Ile Ser Glu Phe GlySer Ala Pro Glu Phe Arg Glu Phe 385 390 395 400 Thr Ile Asp Phe Ser ThrGln Gln Ser Tyr Val Ser Ser Leu Asn Ser 405 410 415 Ile Arg Thr Glu IleSer Thr Pro Leu Glu His Ile Ser Gln Gly Thr 420 425 430 Thr Ser Val SerVal Ile Asn His Thr Pro Pro Gly Ser Tyr Phe Ala 435 440 445 Val Asp IleArg Gly Leu Asp Val Tyr Gln Ala Arg Phe Asp His Leu 450 455 460 Arg LeuIle Ile Glu Gln Asn Asn Leu Tyr Val Ala Gly Phe Val Asn 465 470 475 480Thr Ala Thr Asn Thr Phe Tyr Arg Phe Ser Asp Phe Thr His Ile Ser 485 490495 Val Pro Gly Val Thr Thr Val Ser Met Thr Thr Asp Ser Ser Tyr Thr 500505 510 Thr Leu Gln Arg Val Ala Ala Leu Glu Arg Ser Gly Met Gln Ile Ser515 520 525 Arg His Ser Leu Val Ser Ser Tyr Leu Ala Leu Met Glu Phe SerGly 530 535 540 Asn Thr Met Thr Arg Asp Ala Ser Arg Ala Val Leu Arg PheVal Thr 545 550 555 560 Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile GlnArg Glu Phe Arg 565 570 575 Gln Ala Leu Ser Glu Thr Ala Pro Val Tyr ThrMet Thr Pro Gly Asp 580 585 590 Val Asp Leu Thr Leu Asn Trp Gly Arg IleSer Asn Val Leu Pro Glu 595 600 605 Tyr Arg Gly Glu Asp Gly Val Arg ValGly Arg Ile Ser Phe Asn Asn 610 615 620 Ile Ser Ala Ile Leu Gly Thr ValAla Val Ile Leu Asn Cys His His 625 630 635 640 Gln Gly Ala Arg Ser ValArg Ala Val Asn Glu Glu Ser Gln Pro Glu 645 650 655 Cys Gln Ile Thr GlyAsp Arg Pro Val Ile Lys Ile Asn Asn Thr Leu 660 665 670 Trp Glu Ser AsnThr Ala Ala Ala Phe Leu Asn Arg Lys Ser Gln Phe 675 680 685 Leu Tyr ThrThr Gly Lys 690

What is claimed is:
 1. A recombinant expression vector, said vectorencoding an affinity tag and protein comprising at least a portion ofbacterial toxin, said toxin selected from the group consisting ofEscherichia coli type 1 verotoxin and Escherichia coli type 2 verotoxin,and wherein said vector comprises nucleic acid encoding at least aportion of an amino acid sequence selected from the group consisting ofSEQ ID NOS:3, 8, 21, 23, 25, 27, 47 and
 49. 2. The recombinantexpression vector of claim 1, wherein said affinity tag comprises apolyhistidine tract.
 3. The recombinant expression vector of claim 1,wherein said affinity tag comprises the maltose binding protein.
 4. Ahost cell capable of expressing a recombinant verotoxin protein as asoluble protein at a level of at least 1 milligram per 1 OD of said hostcells per liter.
 5. The host cell of claim 4, wherein said recombinantverotoxin protein is expressed as a soluble protein at a level of atleast 10 milligrams per 1 OD of said host cells per liter.
 6. A hostcell containing a recombinant expression vector, said vector encoding anaffinity tag and protein comprising at least a portion of bacterialtoxin, said toxin selected from the group consisting of Escherichia colitype 1 verotoxin, Escherichia coli type 2 verotoxin, and Shiga toxin,wherein said expression vector selected from the group consisting ofpET24hisVT1 BL+, pET24T7VT1 B, pET24hisVT2BL+, and pET23hisVT2B.
 7. Thehost cell of claim 6, wherein said host cell is a bacterial cell.
 8. Thehost cell of claim 7, wherein said host cell is an Escherichia colicell.
 9. The host cell of claim 7, wherein said host cell is a Shigellacell.
 10. The host cell of claim 6, wherein said host cell is aneukaryotic cell.
 11. The host cell of claim 10 is an insect cell. 12.The host cell of claim 10, wherein said host cell is a mammalian cell.13. A method of generating neutralizing antibody directed againstEscherichia coli verotoxin comprising: a) providing in any order: i) anantigen comprising a fusion protein comprising a non-toxin proteinsequence containing a histidine tract, and at least a portion of aEscherichia coli verotoxin, said toxin selected from the groupconsisting of type 1 toxin and type 2 toxin, and ii) a host; and b)immunizing said host with said antigen so as to generate a neutralizingantibody.
 14. The method of claim 13, wherein said antigen furthercomprises a fusion protein comprising a non-toxin protein sequence andat least a portion of Escherichia coli verotoxin selected from the groupcomprising Escherichia coli type 1 verotoxin and Escherichia coli type 2verotoxin.
 15. The method of claim 13, wherein said antigen iscross-linked.
 16. The method of claim 13, wherein said host is achicken.
 17. The method of claim 13, wherein said host is a mammal. 18.The method of claim 17, wherein said mammal is a human.
 19. The methodof claim 13, further comprising step c) collecting said antibodies fromsaid host.
 20. The method of claim 19, further comprising step d)purifying said antibodies to provide an antibody preparation.
 21. Themethod of claim 20, wherein said purifying comprises affinitypurification.
 22. The antibody raised according to the method of claim13.
 23. The antibody raised according to the method of claim 22, whereinsaid antibody is an avian antibody.
 24. The antibody raised according tothe method of claim 13, wherein said antibody is protective.
 25. Amethod of treatment comprising: a) providing: i) neutralizing antitoxindirected against at least a portion of an Escherichia coli recombinantverotoxin in an aqueous solution in therapeutic amount that isadministrable, and ii) an intoxicated subject; and b) administering saidantitoxin to said subject.
 26. The method of claim 25, wherein saidantitoxin is an avian antitoxin.
 27. The method of claim 25, whereinsaid antitoxin is a mammalian antitoxin.
 28. The method of claim 25,wherein said recombinant Escherichia coli verotoxin is a fusion proteincomprising a non-verotoxin protein sequence and a portion of theEscherichia coli verotoxin VT1 sequence.
 29. The method of claim 28,wherein said recombinant Escherichia coli verotoxin is a fusion proteincomprising a non-verotoxin protein sequence and a portion of theEscherichia coli verotoxin VT1 subunit A sequence.
 30. The method ofclaim 28, wherein said recombinant Escherichia coli verotoxin is afusion protein comprising a non-verotoxin protein sequence and a portionof the Escherichia coli verotoxin VT1 subunit B sequence
 31. The methodof claim 25, wherein said recombinant Escherichia coli verotoxin is afusion protein comprising a non-verotoxin protein sequence and a portionof the Escherichia coli verotoxin VT2 sequence.
 32. The method of claim31, wherein said recombinant Escherichia coli verotoxin is a fusionprotein comprising a non-verotoxin protein sequence and a portion of theEscherichia coli verotoxin VT2 subunit A sequence.
 33. The method ofclaim 31, wherein said recombinant Escherichia coli verotoxin is afusion protein comprising a non-verotoxin protein sequence, and aportion of the Escherichia coli verotoxin VT2 subunit B sequence. 34.The method of claim 25, wherein said recombinant Escherichia coliverotoxin is a fusion protein is cross-linked.
 35. The method of claim25, wherein said subject is an adult.
 36. The method of claim 25,wherein said subject is a child.
 37. The method of claim 25, whereinsaid administering is parenteral.
 38. The method of claim 25, whereinsaid administering is oral.
 39. A method of prophylactic treatmentcomprising: a) providing: i) a neutralizing antitoxin directed againstat least one Escherichia coli recombinant verotoxin in an aqueoussolution in therapeutic amount that is parenterally administrable, andii) at least one subject is at risk of diarrheal disease; and b)parenterally administering said antitoxin to said subject.
 40. Themethod of claim 39, wherein said antitoxin directed against saidEscherichia coli recombinant verotoxin is directed against Escherichiacoli verotoxin type
 1. 41. The method of claim 39, wherein saidantitoxin directed against said Escherichia coli recombinant verotoxinis directed against Escherichia coli verotoxin type
 2. 42. The method ofclaim 40, wherein said antitoxin is directed against said Escherichiacoli recombinant verotoxin is directed against Escherichia coliverotoxin type 1 subunit B.
 43. The method of claim 41, wherein saidantitoxin is directed against said Escherichia coli recombinantverotoxin is directed against Escherichia coli verotoxin type 2 subunitB.
 44. The method of claim 39, wherein said subject is at risk ofdeveloping extra-intestinal complications of Escherichia coli infection.45. The method of claim 39, wherein said extra-intestinal complicationis hemolytic uremic syndrome.
 46. A vaccine comprising a fusion protein,said fusion protein comprising a non-toxin protein sequence and at leasta portion of a bacterial toxin, said verotoxin selected from the groupconsisting of Escherichia coli type 1 verotoxin, Escherichia coli type 2verotoxin, and Shiga toxin.
 47. The vaccine of claim 46, furthercomprising a fusion protein comprising a non-toxin protein sequence andat least a portion of Escherichia coli verotoxin type 1 verotoxin. 48.The vaccine of claim 47, further comprising a fusion protein comprisinga non-toxin protein sequence and at least a portion of Escherichia coliverotoxin type 2 verotoxin.
 49. The vaccine of- claim 47, wherein saidnon-toxin protein sequence is selected from the group consisting ofpoly-histidine tract and maltose binding protein.
 50. The vaccine ofclaim 47, wherein said vaccine is substantially endotoxin-free.
 51. Thevaccine of claim 47, wherein said bacterial toxin is cross-linked.